A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells

Abstract

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.

FIG. 1.

Morphological aspect of isolated human fetal liver stem cells (hFLSCs). Trypan Blue dye exclusion test of hFLSCs (A, magnification 200×); hFLSCs at first stage (B, magnification 200×; C, 400×); The nuclei are stained blue (DAPI 4,6-diamidino-2-phenylindole) in the DAPT assay. The merge vision represents positive cells. hFLSCs form colonies during proliferation (D, 400×; E, 200×); hFLSCs at confluence (F, 200×). Scale bars, 50 μm (A, B, E, and F); 100 μm (C and D). Color images available online at www.liebertpub.com/rej

FIG. 2.

Cell growth curve of isolated human fetal liver stem cells (hFLSCs). Initially, 300,000 cells were plated in one of the six-well dish and expanded in Dulbecco's modified Eagle medium (DMEM)/F12. Cells were sub-cultured and counted every 3–4 days. The total number of the cells is reported on the graph. (▪) Freshly cultured cells; (●) cryopreserved cells.

FIG. 3.

Characterization of human fetal liver stem cells (hFLSCs) by immunohistochemistry (A–F) and immunofluorescence (G, H). hFLSCs are positive for fetal liver-specific marker alpha-fetoprotein (AFP) (A, magnification 400×; brown 3,3′-diaminobenzidine [DAB] stain), stem cell marker OV-6 (B, magnification 400×; counterstained with Hematoxylin), epithelial markers such as CK18 (C, 400×; red 3-amino-9-ethylcarbazole [AEC] stain) and CK19 (D, 200×; red AEC stain), and fetal liver stem cell marker CD326 (E, magnification 200×; red AEC stain). Negative control (F, 200×). They are also positive for stem cell marker c-Kit (G, magnification 400×), nucleus of cells were observed by 4′,6-diamidino-2-phenylindole (DAPI) staining (H, mangification 400×). Scale bars, 50 μm (D, E, and F); 100 μm (A, B, C, G, and H). Color images available online at www.liebertpub.com/rej

FIG. 4.

Phenotyping profiles of human fetal liver stem cells (hFLSCs) obtained by the collagen perfusion, gravity sedimentation, and Percoll density gradient centrifugation (CSP) method and magnetic-activated cell sorting (MACS) method from passage 1 (P1) to P20. Nine kinds of surface markers including epithelial cell adhesion molecule (EpCAM), c-kit, CD44, OV-6, CD81, alpha-fetoprotein (AFP), CK19, CK18, and albumin (ALB) are represented from A to I. (ART) CSP method; (ART) MACS method. () Significant statistical difference (p<0.05) of the expression of cell-surface markers for these two methods.

FIG. 5.

Human fetal liver stem cells (hFLSCs) were differentiated to hepatocytes. When cultured with 10 ng/mL fibroblast growth factor 4 (FGF4), hFLSCs flatten and increase in size to become polygonal cells characteristic of hepatocytes (A, magnification 400×), differentiated hepatocytes express glycogen deposits, which stain pink with Periodic Acid Schiff (PAS) (B, magnification 400×, negative control; C, magnification 400×; D, magnification 200×). (E–H) Effect of human growth factor (HGF) and FGF4 on the activity of glucose 6-phosphatase (G6Pase), which is shown as the brown–black precipitates (E, magnification 400×, negative control; F, magnification 400×, 12 days after HGF and FGF4 treatment; G and H, magnification 400×30 days after treatment). The Indocyanine Green (ICG) uptake assay illustrated that the cells were able to process the ICG dye (I and J, magnification 400×, the undifferentiated group; K, magnification 400×, ICG uptake cells; L, magnification 400×, ICG excretion after cells cultured 6 hr). (M–R) Fluorescence images showing that mature hepatocytes process low-density lipoprotein (LDL) (1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine perchlorate (Dil)-Ac-labeled LDL [Dil-Ac-LDL], red, magnification 400×). (S and T) A total of 2×10⁶ cells were cultured without fetal bovine serum (FBS) in the presence of 10 ng/mL HGF and FGF4 (●), and cells were cultured in the absence of growth factors (▪). Scale bar, 100 μm except for D (50 μm). DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/rej

FIG. 6.

The human fetal liver stem cells (hFLSCs) were differentiated to cholangiocytes. When cultured in biliary differentiation medium, the cells switched to short spindle, triangular, and round shapes and were similar to biliary epithelial cells cultured in vitro (A–C, magnification 400×). These cells were stained by CK7 (tetramethylrhodamine [TRITC]) (D–F, magnification, 400×) and CK19 (fluorescein isothiocyanate [FITC]) (G–I, magnification 400×) in different weeks (0, 2, 4 weeks). Scale bar, 100 μm. Color images available online at www.liebertpub.com/rej

FIG. 7.

The pluripotency potential of isolated human fetal liver stem cells (hFLSCs). The hFLSCs were differentiated to osteoblasts (A–F), adipocytes (G–I), and endothelial cells (J–N) with different induction media. Bone induction: Differentiated osteoblasts present a longitudinal and interwoven conformation, deposit calcium into the extracellular matrix, which show nacarat mineralized nodes with Alizarin Red stain (A, magnification 400×, negative control; B, magnification 200×; and C, magnification 400×) and show black with von Kossa stain (D, magnfication 400×, negative control; E, magnification 400×); counterstained with 1% Neutral Red for cytoplasm which show red (F, magnification 400×). Adipose tissue induction: Differentiated cells show increased size and accumulate vacuoles that are positive for Oil Red O stain (G, magnification 400×, negative control; H, magnification 400× and I, 400×). Endothelium induction: Differentiated endothelial cells become spindle-shaped with formation of linear channels between cells (J, magnification 400×); more than 90% of cells are positive for CD31 (K, magnification 400×, negative control; L, magnification 400×; red 3-amino-9-ethyl-carbazole [AEC] stain) and Factor VIII-related antigen (M, magnification 400×, negative control; N, magnification 400×; fluorescein isothiocyanate [FITC] stain). Scale bar, 100 μm except for B (50 μm). Color images available online at www.liebertpub.com/rej