Human fetal liver (FL) tissues were obtained from aborted fetuses at 13-20 wk gestation in accordance with the Institute’s ethical guidelines. The fetuses were collected within 2 h of abortion. Single cell suspension was prepared from the liver by the two-step collagenase perfusion method and the cells were passed through a 70 μ-nylon mesh. The viability of the cells was assessed by the Trypan blue dye exclusion test. All the donors had been serologically screened for syphilis, toxoplasmosis, rubella, hepatitis B and C, human immunodeficiency virus 1, cytomegalovirus, parvovirus, and herpes simplex types 1 and 2.

Isolated human fetal liver cells were suspended in 1 mL of PBS with 5 mmol/L EDTA and labeled with anti CD34 magnetic labeled beads. CD34+ hepatic cells were separated by magnetic cell sorting (MACS) using the human direct CD34-progenitor kit (Miltenyi Biotech; Germany) according to the manufacturer’s instructions. The flow-through fraction was used as a source of CD34- cells. The enriched cells were analyzed for AFP (hepatic lineage marker) and CD45 (leukocyte lineage marker) using CD34PE/AFP FITC and CD45 PE on Flowcytometer (FACS Caliber Becton & Dickinson USA). To assess the nonspecific binding appropriate isotype controls were included.

Enriched CD34+ positive cells were cultured for a long term duration in a serum free medium. Two million cells were seeded on collagen typeI(Sigma Co.) coated 6 well plates (Nunclon). The medium DMEM (Sigma) was supplemented with 50 mg/L L-glutamine (Sigma Co.), 50 mg/L essential amino acids, penicillin, streptomycin, amphotericin B (Hi Media) and 10% heat inactivated fetal bovine serum (Gibco BRL), 50 ng/mL hepatocyte growth factor (HGF) (Sigma Co.), 20 ng/mL of epidermal growth factor (Gibco) and 10 ng/mL basic fibroblast growth factor (bFGF). Non-adherent cells were removed after 24 h and subsequently the medium was changed daily for 3 d. Cultured cells were analyzed for liver specific genes on the following days of culture: d 0, 7, 14, 21, 28 and 35. Cells were trypsinized with trypsin-EDTA diluted 1:10 in PBS for 5-7 min at 37°C on each occasion. For sub passaging, near-confluent cultures were split 1:3 using half-strength trypsin-EDTA for 6-10 min at 37°C.

The trypsinized cells were collected by washing with phosphate-buffered saline. Smears were prepared using cytospin (Shandon) on glass slides. The smears were fixed with methanol at -20°C for 5 min and acetone at 4°C for 15 s. The smears were washed with PBS and blocked with 1% bovine serum albumin (Sigma Co.) in PBS for one hour at room temperature, cells were incubated with mouse anti human-AFP (Santa Cruz) diluted 1:100 (in PBS) at 4°C for two hours and then stained with FITC conjugated rabbit anti mouse secondary antibody, diluted 1:1000 in PBS at 4°C for 30 min. After the incubation the cells were washed with PBS twice and the cells were analyzed under fluorescence and visible light.

For each slide, four randomly chosen areas were analyzed. The total cell number and the number of positive cells were assessed. For morphologic analysis the cells were split into two categories: small cells were round shaped cells with one nucleus and a small rim of cytoplasm. Large cells showed irregular cell shape with a prominent cytoplasm. For each area the total number of cells and the number of small cells were counted. The ratio of positive to total cells and the ratio of small to total cells were calculated as percentages.

Four million cells from each group were spun at 3000 rpm and 3 mL of 0.2% Triton x-100 was added to the pellet. Cells were lysed by freezing and thawing three times, and then the lysate was spun at 15 000 r/min for 45 min at 4°C. The supernatant was used for the estimation of AFP (Figure 1). 20 μL of the supernatant was used for AFP activity using ELISA in accordance with the protocol provided in the kit (Pathozyme).

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Figure 1 Sorted cells stained with FITC labeled anti human AFP antibody and observed under fluorescent (Zeiss) microscope (× 400). Cells were observed to be positive for AFP and a thin rim of cytoplasm with a big nucleus.

Total RNA was extracted from CD34+ sorted cultured cells using Trizol (Invitrogen Life technology). Liver specific transcripts were analyzed using complementary DNA, which was amplified later for 35 cycles using primer pair specific for AFP, albumin and CK-18. PCR products were separated in 1.5% agarose gel and visualized under UV light. Primer pairs used in the study are listed in Table 1.

Flowcytometer analysis was done with freshly isolated or cultured cells. Cell number was assessed by using flow cytometer (BD). Mean values and standard deviations (SD) of cell numbers were calculated. Statistical analysis was performed using the two way Anova.

The viability of isolated cells ranged from 85%-94%. Primary cell isolation yield increased with increases in gestation (13-20 wk) from 39 ± 2.3 × 10⁶ to 90 ± 3.4 × 10⁶. There was no loss in the viability following magnetic cell sorting; viability ranged from 85%-90%.

Analysis of enriched CD34 positive cells showed two populations of cells CD34+/CD45+ and CD34+/CD45-. The majority of cells were CD34+/CD45-, and the CD 45 marker was used to exclude for lymphoid and myeloid lineage cells. The data revealed that CD34+/CD45+ (CD34 and CD45 positive cells) were 5.5% + 0.56% in group-I, 4.8% + 0.72% in group-II and 4.5% + 0.73% in group-III. Whereas CD34+/CD45- (CD45 negative cells) ranged from 4.1% + 0.82% in group I, 4.2% + 0.9% in group II and 3.9% + 0.8% in group-III, CD 45 + cells ranged from 0.3% to 0.5% (Table 2).

Of the enriched cells, when analyzed after staining with AFP, more than 70% ± 1.5% were observed to be positive for AFP and had a thin rim of cytoplasm with a big nucleus (Figure 1). AFP estimation in CD34 positive enriched cells increased four to five fold in comparison with non-enriched cells. (Table 3, Figure 2).

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Figure 2 The graph shown is representative of the three independent experiments. The activity was expressed in μg/mg protein.

Culture of the CD34 positive cells showed increasing numbers with a significant rise in cell count from 7.4 ± 1.3 × 10⁶/plate at d 0 (Figure 3A and B) to 8.6 ± 1.4 × 10⁶/plate at d 35 of culture (Figure 3C). The cells, which were negative for CD34 when cultured, showed a decrease in cell number from 7.6 ± 0.9 × 10⁶ cells at d 0 to 2.7 ± 0.08 × 10⁶ cells at d 35. At d 28, two populations of cells were found in cultures of CD34+ cells. Cell numbers of cultured CD34+ cells increased significantly (P < 0.05) after d 7 when compared with the numbers of cultured CD34- cells (Figure 4).

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Figure 3 A: The sorted cells on d 0 of culture. Cells were cultured in the long term. The cells are showing intact morphology (× 400); B: The cultured cells on 14th d. The cells are showing intact morphology (× 400); C: The cultured cells on 35th d. The cells are showing intact morphology (× 400).

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Figure 4 Culture of CD34+ sorted primary cells.

To confirm that the sorted cells were of hepatic lineage, cultured cells were further analyzed for the expression of liver specific mRNA transcript from d 0 to d 35 of culture (Figure 5). AFP and CK-18 expression was observed in the sorted culture cells, which revealed that these cells were capable of differentiating into hepatic and biliary lineages, suggesting the cellular bipotency of progenitor cells. Expression of albumin was noted only after 8 d of culture, indicating that the cells were in the process of maturation.

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Figure 5 Gene expression analysis in different days of cultures of hepatic progenitors.

In our study, we found that cells enriched by haematopoietic stem cell marker CD34 expressed hepatocyte specific markers, but the majority of these cells were negative for leukocyte markers CD45. In the present study we have used two markers, AFP (hepatic marker) and CD45 (common leukocyte marker) in order to identify the CD34 positive cells of hepatic lineage. The flowcytometric analysis data showed two populations of cells CD34+/CD45+ and CD34+/CD45-. The majority of cells were CD34+/CD45- and the CD 45 marker was used to exclude the cells of lymphoid and myeloid lineage.

In the present study CD34+ positive cells were separated by MACS from human fetal liver cells. The data generated by magnetic cell sorting of progenitor cell population using CD34 marker increased AFP activity up to four to five fold in enriched cells when compared with the non sorted cells. As AFP is a marker for proliferation, increases in the AFP activity in the enriched population demonstrates that sorted cells have higher proliferation than the unsorted cells. Our findings also correlate with those of Craig et al and Strain et al who sorted hepatic progenitor cell population by labeling the cells with anti-CD34. The present study also revealed that the AFP activity of the cells before and after sorting with CD34 was more in groupI. This indicates that both CD34 in combination with AFP are good markers to enrich hepatic progenitors. Sorted cells were cultured to study the proliferative activity of the cells. There were increases in cell numbers. This shows that the cells negative for CD34 were matured cells and had low proliferative rates. The sorted CD34 positive cells in cultures showed very high proliferative rates, which indicates that the sorted cells are immature, progenitor cells. The AFP activity indicates that these cells belong to the hepatic lineage. The gene expression analysis in cultures on different days was carried out to study the hepatic markers, AFP, albumin and CK-18. The data reveal that the sorted CD34 positive cells showed AFP expression till 35 d of culture, which shows that the cells are in proliferative states even for 35 d. There was no expression of albumin on d 0, as the sorted cells are immature. With increases over time the expression of albumin started as the cells underwent maturation. The CK-18 expression further confirms that the cells are of epithelial lineage. The culture study revealed that the CD34 positive cells were able to proliferate and differentiate into cells of hepatic lineage.

In conclusion, the above study reveals that CD34 is a good marker for enrichment of hepatic progenitor cells from human fetal livers. This was confirmed by the liver specific marker, AFP which increased after sorting, as CD34 sorting leads to the enrichment of the progenitor population. The expression of hepatic markers in cultured cells concludes that the cells were able to proliferate and differentiate for several days.