A Novel Technique for Hepatic Progenitor Cell Isolation From Normal Adult Rat Livers

Abstract

The transplantation of hepatic progenitor cells (HPCs) is a promising alternate approach to liver transplantation for patients with end-stage liver disease. Here, we report a novel technique for HPCs isolation from normal adult rat livers (no preexposure to chemicals and no injury). HPCs were isolated from normal adult rat livers using a novel four-step collagenase perfusion method followed by density gradient centrifugation. The phenotypic properties of HPCs were characterized by morphological observation, reverse-transcription polymerase chain reaction (RT-PCR), and immunocytochemistry. The results showed that HPCs formed loose colonies and possessed a round or oval shape at culture day 3. These cells proliferated slowly and exhibited progenitor-like characteristics during the 30-day culture period. RT-PCR analysis indicated that the cultured cells were positive for several HPC-specific genes, such as albumin (ALB), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), cytokeratin 19 (CK19), CD45, hepatocyte nuclear factor 1-alpha (HNF-1α), hepatocyte nuclear factor 4-alpha (HNF-4α), and Thy-1. Immunocytochemical staining showed that these cells were consistently positive for ALB, AFP, CK18, CK19, Thy-1, and OV-6. HPCs can be isolated from normal adult rat livers using a simple and effective technique involving four-step collagenase perfusion, further confirming their potential as a strong candidate for hepatocyte therapy.

Acute liver failure is a rare but life-threatening condition that can be caused by various kinds of chronic liver disease or that can arise with no history of liver disease.¹

Liver transplantation is thought to be the only therapeutic option for patients with end-stage liver disease.^2,3 However, the feasibility of liver transplantation is limited by its high cost, a shortage of donor organs, and the need for immunosuppression. Cell transplantation with stem cells could potentially replace liver transplantation in patients with end-stage liver diseases.⁴ Thus, a good deal of research has turned to cell transplantation, such as the transplantation of mature cells or stem/progenitor cells and the potential transplantation of xenogeneic organs and cells.^5–8 As many diseases result from hepatocyte dysfunction, transplantation of hepatocytes or hepatocyte-like cells is a promising alternative for the treatment of acute and chronic liver failure.^9,10 However, this approach is also restricted by a shortage of donor cells and by immunological barriers. Hepatic stem/progenitor cells have been shown to exist in adult rodent and human livers, as demonstrated by the coexpression of stem cell and hepatic lineage markers.^11,12 However, in adult normal livers, stem cells are quiescent, existing only in low numbers around the periportal region, and they proliferate only following severe, prolonged liver trauma.¹³ There are a variety of methods to isolate these stem cells. Animal models, such as diethylnitrosamine-treated rats, the 2-acetylaminofluorene/partial hepatectomy model, and the retrorsine/partial hepatectomy model have been used.^14–17 The reagents used in these models are different, but the time to induce liver disease in these animal models is uniform. The purpose of these methods is the same: that is to cause severe, prolonged liver trauma. Although a great deal of stem cells can be isolated from these disease model animals, the cells acquired through these methods are taken from abnormal organisms. Another hypothesis is that stem cells work throughout the development of hepatocellular carcinoma and might play important roles in this process.^18–21 Cells can be separated from the liver, but their clinical application is also dependent on the availability of high-quality donor livers.⁶ Therefore, using noninjured livers, we tried to isolate a new population of progenitor cells similar to oval cells, which can proliferate and renew and have the ability to differentiate into hepatocytes, that can be used to replace dysfunctional livers. Most of such research have focused on the analysis of cell proliferation in vivo, and only seldom do experiments showing cell proliferation in vitro for more than 20 days. Here, we isolated a population of stem cells exhibiting similarities to hepatocytes and stem or progenitor cells from normal adult rats. These cells were characterized by immunocytochemical analysis and reverse-transcription polymerase chain reaction (RT-PCR) techniques.

Materials and Methods

Chemicals and Reagents

Chemicals for cell isolation and culture were purchased from GIBCO Corporation. Dulbecco’s modified Eagle’s medium (DMEM), trypsin–ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS), and Trizol Reagent were purchased from GIBCO Corporation; bovine serum albumin and type collagenase IV were from Sigma Chemical Corporation (St. Louis, MO); and dispase and DNase were from Roche Corporation. Percoll PLUS reagent was purchased from GE Corporation. Regular culture dishes and flasks were purchased from BD Falcon (Franklin Lakes, NJ). Other reagents for RT-PCR were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Animals and Treatments

The male Sprague Dawley rats used in our studies were obtained from the First Affiliated Hospital, Zhejiang University. All rats were housed in a climate-controlled (20°C) room with a 12-hour light/dark cycle. All experimental protocols and study methods were approved by the Animal Care Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University.

Isolation of Hepatic Progenitor Cells

Primary hepatic progenitor cells (HPCs) were isolated from normal Sprague Dawley rats weighing 100–150 g using a modification of a two-step collagenase perfusion method that has been described previously.^22,23 This modified method contains four steps.²⁴ First, the perfusate were prepared and placed in 39°C water bath. Rats were anesthetized using 4% chloral hydrate at the dose of 0.7 ml/100 g. The rats were fixed on the operation panel after disinfection. Under strict aseptic technique, the abdominal cavity was opened from the ventral midline, and the portal vein was exposed. The ligaments were separated around the liver. Then the portal vein was punctured by puncture needle designed with a pipeline connecting the portal vein puncture needle. The needle was fixed with a vascular clamp at the portal vein and then the inferior vena cava was cut. For the first step of perfusion, using the peristaltic pump, the liver was perfused with EDTA/D-Hanks solution, which was preheated to the temperature 39°C, and the speed of micropump was regulated at 50 ml/min. Total volume of first perfusion solution was 150 ml. The second step of perfusion involved the use of D-Hanks solution. Total volume was 50 ml. After the first two-step in situ perfusion, the liver was removed to the stainless steel container, with the portal vein fixed with the puncture needle. The third-step perfusion was performed with dispase/D-Hanks solution, and the volume was 50 ml. Then, the perfusate was discarded. The fourth-step perfusion used collagenase IV/Hanks solution of 50 ml. After perfusion, the hepatic peplos was teared sharply, and the fibrous connective tissue was removed in an ice bath under 200-mesh stainless steel sieve with filtered liver cells. The supernatant was harvested and centrifuged at 4°C three times at 150g for 3 minutes with PBS. The nonparenchymal cells were harvested in pellets, and density gradient centrifugation was performed to separate HPCs. Subsequently, the cells was suspend with 2 ml WBD (MgCI₂ ? 6H₂O, 0.0159 g; MgSO₄ ? 7H₂O, 0.0159 g; DNase, 0.0159 g; and ultrapure water, 150 ml), and the suspension was loaded onto a discontinuous gradient of 50%, 70%, and 90% Percoll and centrifuged at 350g for 25 minutes. The cells between the 70% and 90% Percoll layers were harvested, washed with PBS, and centrifuged twice at 150g for 3 minutes at 4°C. Then, the HPCs suspension was resuspended and plated at a density of 4 × 10⁴ cells ? cm⁻² in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were cultured at 37°C, in a 5% CO₂ atmosphere with 100% humidity. The culture medium was replaced 2 days after plating and every 2 days after that. About 21 days later, HPCs were harvested for RT-PCR and immunocytochemical analysis. Cell morphology was observed under phase-contrast microscopy during the entire culturing period.

Reverse-Transcription Polymerase Chain Reaction

To determine the molecular phenotypes of HPCs, HPC-specific gene expression was analyzed by RT-PCR as described previously.²⁵ Briefly, total RNA was extracted from cultured HPCs and hepatocytes using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was reverse-transcribed, and cDNA was synthesized using QuantiTect reverse transcription reagent (Qiagen, Valencia, CA). The reverse-transcribed cDNA was amplified by PCR using HPC-specific gene primers (Table 1). Glyceraldehyde phosphate dehydrogenase was used as an internal control.

Table 1: Primers Used for HPC Gene Amplification

Immunocytochemistry

The liver stem cell-specific and hepatic-specific markers (detected with mouse monoclonal antibodies, dilutions in parentheses) OV-6 (1:100), alpha-fetoprotein (AFP; 1:200), Thy-1 (1:100), cytokeratin 18 (CK18; 1:100), albumin (ALB; 1:100), and cytokeratin 19 (CK19; 1:200) were used to characterize the cultured HPCs. All antibodies were purchased from Santa Cruz Biotechnology. The HRP-streptavidin reagent (Santa Cruz, CA) was used on cultured cells that were fixed and stained directly in the culture dishes. The immunocytochemical staining was performed as described previously.²⁶ Briefly, the cultured cells were rinsed three times with PBS, fixed with 4% paraformaldehyde solution for 15 minutes at room temperature, washed once with PBS, and permeabilized with 80% methanol for 15 minutes at−20°C. Following a PBS wash, the cells were incubated with blocking buffer (PBS with 5% FBS) for 60 minutes at room temperature. Subsequently, the cells were incubated overnight with primary antibodies, followed by incubation with biotinylated polyclonal anti-mouse and anti-goat or anti-rabbit IgG. Between each antibody incubation step, the cells were washed three times with PBS. Digital images of the cells were taken under light microscopy.

Results

Morphology of HPCs

The total number of HPCs separated from each rat weighing approximately 100 g was 2.5 ± 0.5 × 10⁵. The viability which was tested by trypan blue viability test was nearly 99% in the freshly isolated cells. The freshly isolated HPCs attached to the culture plates within 48 hours. Some small scatter clones were observed after 3 days of culturing. These cells proliferated slowly and exhibited progenitor-like characteristics, forming sharply bordered colonies of densely packed small cells with a low cytoplasmic/nuclear ratio after 7 days of culturing (Figure 1).

Figure 1: Morphology of cultured hepatic progenitor cells (HPCs). Phase contrast images show cultured cells at days 7 (A–C), 10 (D–F), 14 (G–I), and 21 (J–L). Single HPCs rapidly proliferated to form colonies by day 7. The cells formed sharply bordered colonies and exhibited a round to oval shape with a low cytoplasmic/nuclear ratio typical of stem cells after 10 days in culture. Original magnification: A, D, G, and J, 10×; B, E, H, and K, 20×; and C, F, I, and L, 40×.

Results of RT-PCR

The RT-PCR results showed that the cultured cells were positive for several genes characteristically expressed by stem cells, hepatocytes, biliary epithelial cells, and nonparenchymal cells (Figure 2). Strong expression of Thy1 and CK18 was observed in addition to intermediate expression levels of ALB, AFP, CK19, CD45, hepatocyte nuclear factor 1-alpha (HNF-1α), and hepatocyte nuclear factor 4-alpha (HNF-4α). The HPCs were negative for TDO2 and TAT expression.

Figure 2: Reverse-transcription polymerase chain reaction for liver stem cell mRNA. Lane 1 cells were hepatic progenitor cells, which were cultured in vitro in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 21 days. Primary hepatocytes (lane 2) were used as a control. Lane 3: DNA marker.

Immunocytochemistry

Immunocytochemistry was performed to characterize the HPCs. The immunocytochemical results demonstrated that the cultured cells were positive for OV-6, Thy1, AFP, CK18, and ALB and for CK19 (Figure 3).

Figure 3: Results of immunocytochemical analysis. Immunocytochemical staining showed that the cultured cells were consistently positive for the HPCS markers OV-6 (A), Thy-1 (B), ALB (C), and AFP (D). These cells were also positive for CK18 (E) and CK19 (F), which are expressed by hepatocytes and biliary epithelium, respectively. Original magnification: A–E, 40×; F, 20×.

Discussion

HPCs may exist in or near the portal area, but it is hard to isolate and identify HPCs in normal livers.^26,27 Many studies have isolated hepatic stem cells or oval cells from liver injury models induced by different chemicals, such as allyl alcohol,²⁶ carbon tetrachloride, and D-galactosamine.^28–30 These models often result in chromosomal abnormalities in hepatic stem cells, which preclude their use in clinical settings.⁶ High-quality and phenotypically normal HPCs are very important for cytotherapy (hepatocyte therapy) of liver diseases. Therefore, in this study, we isolated HPCs from unmanipulated adult rats. The conventional method, which involves two-step collagenase perfusion, is commonly used for hepatocyte isolation, both from animal models and humans. However, the yield and viability of freshly isolated hepatocytes are relatively low. Our previous results showed that four-step liver perfusion with EDTA, dispase, collagenase, and DNase allows the isolation of a greater number of pure and viable hepatocytes from normal human livers than that using the conventional methods.²⁴

In this study, the four-step liver perfusion protocol was first applied to isolate HPCs from unmanipulated adult rat livers. Through the four-step perfusion method, based on the two-step perfusion method, combined with density gradient centrifugation, high-quality liver progenitor cells can be obtained. And these cells can also be cultured for short-term, which provides a new way. Our studies show that four-step perfusion of the liver cells from liver stem/progenitor cells has the following advantages: 1) during the four-step perfusion method, first step of the program was the puncture of portal vein, and so the following solution was perfused from the portal vein, which makes the perfusion more sufficient; 2) the first step of in situ perfusion involved the use of EDTA, which can chelate extracellular calcium, loose the connections between the cells, and can remove most of blood cells, and hence the perfusion become more complete; 3) the collagenase owns the characteristics of calcium-dependent. In two-step perfusion method, the collagenase perfusion was close behind the EDTA perfusion, which can reduce the effect of collagenase digestion, and so we add the two steps, which contains D-Hanks perfusion and dispase perfusion, not only to remove the EDTA which is in blood vessels but also to bring into full play synergistically to improve the effect of collagenase; 4) the collagenase IV can digest the matrix components of the liver, thereby undermining the close connections between cells and gap junctions. Water bath temperature raised to 39°C, which ensures the optimum temperature of collagenase perfusion of 37°C, to obtain a good separation; and 5) in the subsequent washing process, washing buffer containing DNase, which can prevent cells from gathering into groups, will improve cell viability and yield. The study further confirms the use of the four-step method that has been performed in our laboratory.

Percoll solution does not penetrate into the biofilm, and Percoll have the characters of nonvenomous, low osmotic pressure, and low diffusion constant, which also can form a stable gradient of density characteristics. Based on the different density of liver progenitor cells and liver cells, after centrifugation, liver stem/progenitor cells can reside in a specific density range. After density gradient centrifugation, the cells below 1.035 g/ml were cell debris, between 1.070 and 1.110 g/ml were Kupffer cells, endothelial cells, stellate cells, and liver cells. We chose the Percoll solution of 50%, 70%, and 90% which had density 1.067, 1.090, and 1.12 g/ml, respectively. The cells with density between 1.090 and 1.12 g/ml had a high production, good activity, uniform cell size, and similar shape.

This method does not require preinduction of damage by chemicals or toxins. The number of freshly harvested HPCs reached 2.5 ± 0.5 × 10⁵. We did not add insulin, HGF, and L-ascorbic acid into the medium. But the FBS contains a variety of plasma proteins, peptides, fat, carbohydrates, growth factors, hormones, inorganic substances, and these substances can maintain the physical balance of the cells.

Small HPC clones were observed after 3 days of culturing. These cells proliferated slowly and exhibited morphological characteristics of HPCs after 7 days of culturing, showing specific surface marker for liver cell such as ALB, AFP, CK18, HNF-1a, and HNF-4a; bile duct epithelial cell surface marker such as CK19; and the hematopoietic stem cell surface marker Thy-1, CD45, and oval cell surface marker OV-6. Thus, the phenotype of HPCs showed some similar characteristics to that of oval cells. Oval cell is the expertised stem cell in liver, which owns the oval shape, and is capable of self-renewal and multipotent differentiation. The cells owns several abilities, such as its oval shape, the ability to express OV-6 and to renew itself, so the cells are similar to oval cells, may be oval cells or downstream of oval cells. Not yet draw a conclusion.

Number of freshly isolated cells is 2.5 ± 0.5 × 10⁵/100 g, and the yield is not high enough. This is difficult to meet the needs of bioartificial liver and liver cell transplantation for treatment of end-stage liver disease, and so the liver progenitor cells need to be cultured in vitro to achieve the required amount.

Although the cells showed a good proliferation during culture, the cells change its shape and slow down the proliferation. That may be due to nutritional deficiency or stimulation of trypsin. The cell proliferation in vitro needs to be further studied. We performed 50 separations, and results were similar, but the statistical analysis was not made. Therefore, the culture condition of the liver progenitor cells in vitro remains to be studied further.

Conclusion

HPCs can be isolated from normal adult rat livers using a simple and effective technique involving four-step collagenase perfusion, confirming the potential of these cells to be as a potential candidate for hepatocyte therapy.