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Stem Cells 2003;21:449-458 www.StemCells.com
© 2003 AlphaMed Press

Hepatic Progenitor Cell Lines from Allyl Alcohol-Treated Adult Rats Are Derived from {gamma}-Irradiated Mouse STO Cells

Mingjun Zhanga,c, Stewart Sellc, Hyam L. Lefferta,b

a Department of Pharmacology and
b Center for Molecular Genetics, University of California, San Diego, La Jolla, California, USA
c Division of Experimental Pathology, Albany Medical College, Albany, New York, USA

Key Words. STO cell feeder layers • Radiation artifacts

Hyam L. Leffert, M.D., Department of Pharmacology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0636, USA. Telephone: 858-534-2354; Fax: 858-534-6833; e-mail: hleffert{at}ucsd.edu website: http://medicine.ucsd.edu/pharmaco/hlleffert.html


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In attempts to recharacterize several markers of putative rat liver progenitor cells, single-stage reverse transcription-polymerase chain reaction (RT-PCR) analyses failed to confirm the reported immunochemical detection of albumin, {alpha}1-fetoprotein, and cytochrome P450-1A2 in the clonal line, 3(8)#21, and the cloned derivative, 3(8)#21-EGFP (enhanced green fluorescent protein). Undetectable expression occurred whether or not both lines were cultured on or off feeder layers of {gamma}-irradiated mouse embryonic STO (SIM [Sandoz inbred Swiss mouse] thioguanine-resistant ouabain-resistant) cells. PCR amplification of liver progenitor cell chromosomal (rat and mouse Pigr, rat INS1, mouse INS2) and mitochondrial (rat and mouse COX1) genes revealed only mouse sequences. Further analyses of rat and mouse COX1 sequences in cells from untampered storage vials of all 11 reported liver progenitor cell lines and strains revealed only mouse sequences. In addition, uniquely similar metaphase spreads were observed in STO, 3(8)#21, and 3(8)#21-EGFP cells. The combined results suggest that the previously reported "rat" liver progenitor cell lines were most likely generated during early derivation in cell culture from {gamma}-radiation-resistant or ineffectively irradiated mouse STO cells used as the feeder layers. These findings reveal new types of artifacts encountered in cocultures of tissue progenitor cells and feeder layer cell lines, and they sound a cautionary note: phenotypic and genotypic properties of feeder layers should be well-characterized before and during coculture with newly derived stem cells and clonal derivatives.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Normal liver progenitor cells (LPCs) have been isolated and studied in vitro by many investigators [1]. Successful plating, function, and plasticity in several systems [2–4] reportedly depend upon the coculture of LPCs with growth-inhibited feeder layers of mouse embryonic STO cells (SIM [Sandoz inbred Swiss mouse] thioguanine-resistant ouabain-resistant cells of E15-17 origin [5]). Mitomycin-C-treated STO feeder layers were first used in 1975 to establish embryonic stem cell lines from mice [6]. STO cells have never been well-characterized, yet they are used extensively; {gamma}-radiation is the current method of choice to inhibit their proliferation [7].

While extending in vitro studies of plasticity in several LPC lines obtained from adult rats injured with allyl alcohol [4], we noticed reports of spontaneous fusion between pluripotent embryonic feeder layer cells and neuronal [8] or bone marrow stem cells [9]. These reports suggested that cell fusion phenomena might underlie some observations attributed to intrinsic plasticities of stem or tissue progenitor cells in other systems. For this reason, and because of the unexpected anomalies described below, we decided to validate the species of origin of 11 recently described rat LPC lines and strains, the cells of which had been cocultured with {gamma}-irradiated mouse STO cells [4]. In particular, we focused on LPC line 3(8)#21 and its derivative, 3(8)#21-EGFP (enhanced green fluorescent protein), which, compared with STO cells ({alpha}1-fetoprotein [AFP-], albumin [ALB-], cytokeratin [CK14-], c-kit-, CD34-, desmin-), reportedly expressed culture-dependent properties of hematopoietic stem cells (CD34+, Thy1.1+, CD45+), hepatic oval cells (AFP+, ALB+, CK14+, c-kit+, OC10+, OV1+, OV6+), bile-duct-like cells (BD1+, structural morphology), and differentiated hepatocytes (H4+, cytochrome P450-1A2+, hepatocyte-like morphology [4]).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture, {gamma}-Irradiation, and Chromosome Analyses
Unless noted, the LPCs were obtained from thawed vials of passage number P19 cells frozen in liquid nitrogen ("P" denotes passage number) and cultured as described elsewhere [4]. Human 293, STO [7], and rat 7777 hepatoma cells were cultured by standard procedures [10]. Subconfluent STO cells were exposed to 5,000 rads (2 Gy/minute) at the Albany Medical College (AMC) using an Isomedix Gammator B. At the University of California, San Diego (UCSD), log-phase STO cells were exposed to 5,300 rads (8 Gy/minute) using a J.L. Shepherd Model 30 Mark 1 {gamma}-irradiator (http://hps.org/aboutthesociety/affiliates/affiliate68.html), calibrated by Fricke dosimetry. STON+ cells (unirradiated neomycin-resistant STO cells, resistant to killing by G418) were constructed from STO cells and {gamma}-irradiated at the AMC. To construct 3(8)#21-EGFP cells, irradiated STON+ cells were cocultured with 3(8)#21 cells. The cocultures were transduced with centrifuged filtered fluids carrying retroviral vector pLNSX/SV/GFP; neomycin-resistant EGFP+ cells were selected and cloned [4]. G-bands by trypsin using giemsa (GTG)-banding was performed by standard procedures [11].

Western Blots
Cells were trypsinized by standard procedures, washed, and centrifuged six times in Na+ phosphate-buffered saline (PBS, 0.01 M, pH 7.4) at 21°C and titered with a Z1 Coulter® Particle Counter (Beckman Coulter; Miami, FL; http://www.beckman.com). Ten million pelleted cells were resuspended in a 0.6-ml preparation of modified radioimmunoprecipitation buffer [12] containing fresh protease inhibitor (Roche; Nutley, NJ; http://www.roche.com) at 4°C. The lysate was passed through a 21-gauge needle; 10 µl of 10 mg phenylmethylsulfonyl fluoride/ml were added to the final solution, which was incubated for 30–60 minutes on ice and centrifuged at 10,000 x g for 10 minutes at 4°C. Protein concentrations were measured in the resulting supernatants using the Coomassie Plus Protein Assay Reagent (Pierce; Rockford, IL; http://www.piercenet.com). Cell lysates (25 µg proteins) were mixed with equal volumes of 2X SDS-PAGE sample buffer (100 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.1% bromophenol blue, 10% ß-mercaptoethanol), boiled for 5 minutes, cooled on ice, and centrifuged for 15 seconds. Electrophoresis was performed on 8% gels at constant voltage (80 v) for 2 hours at 21°C. The separated proteins were transferred onto nitrocellulose membranes (Protran, Schleicher & Schuell; Keene, NH) with a TE 50 Transphor machine (Hoefer Scientific; San Francisco, CA) at constant voltage (70 v) for 16 hours at 4°C.

Immunoblotting was performed with polyclonal rabbit anti-rat albumin (ALB; ICN; Aurora, OH; http://www.icnbiomed.com) and goat anti-rabbit IgG-alkaline phosphatase (AP) (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.scbt.com), or polyclonal goat anti-rat AFP [13] and bovine anti-goat IgG-AP (Santa Cruz Biotechnology). Incubation and washing steps were performed on a rotary shaker at 60–80 rpm. The blotted membranes were washed with tris-buffered saline (TBS; 20 mM Tris-HCl, 500 mM NaCl, pH 7.5) for 15 minutes at 21°C; the TBS was removed and the membranes were blocked in 3% fish gelatin (Sigma; St. Louis, MO; http://www.sigmaaldrich.com)/TBS overnight at 4°C. The blocking solution was removed, and the membranes were washed three times in tris-tween buffered saline (TTBS; 20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5) and incubated in dilute solutions (1% gelatin/TTBS) of primary antibody overnight at 4°C: ALB, 1:200; AFP, 1:1000. The solutions were removed and the blots were washed three times in TBS and incubated in the corresponding dilute (1:1,000) secondary antibody solutions (1% gelatin/TTBS) for 1 hour at 21°C. The solutions were removed, and the blots were washed three times in TTBS and once in TBS, and developed with Western Blue stabilized substrate for alkaline phosphatase (Promega; Madison, WI; http://www.promega.com).

Immunofluorescence and Epifluorescence
Immunofluorescence was performed with cultured cells using either rabbit anti-rat ALB or polyclonal goat anti-rat AFP and Alexa Fluor 546 goat anti-rabbit or donkey anti-goat IgG (Molecular Probes; Eugene, OR; http://www.probes.com). Forty thousand LPCs or 293 cells were plated into 35-mm glass-bottom plastic dishes (MatTek; Ashland, MA; http://www.mattek.com) containing 2 ml medium. Log- and stationary-phase cells were examined 2 and 7 days post-plating. The media were aspirated and the dishes were washed six times in PBS and dried in air. Dried monolayers were fixed with 95% ethanol at 4°C for 1 hour, dried in air, rehydrated in 2 ml PBS for 10 minutes at 21°C (or stored at -20°C and then rehydrated), and washed with 2 ml of 0.1% saponin in PBS for 30 minutes at 21°C. The detergent was aspirated and the dishes were washed three times with 2 ml PBS. The PBS was aspirated, and the cells were blocked in 2 ml of a solution of 5% fish gelatin in PBS for 15 minutes at 21°C. The blocking solution was aspirated, and the dishes were washed with PBS and incubated with primary antibodies in PBS overnight at 4°C using the following dilutions: rabbit anti-mouse ALB (1:200) and goat anti-rat AFP (1:1000). The fluids were aspirated. The dishes were washed with PBS and incubated in dilute solutions (1:75) of secondary antibody for 1 hour at 21°C. The solutions were aspirated, and the dishes were washed with PBS and mounted under 25-mm coverslips (Fisher Scientific; Pittsburgh, PA; https://www1.fishersci.com/index.jsp) in 90% glycerol:10% PBS. Epifluorescence was monitored at {lambda}excitation 546 nm, {lambda}emission 535–550 nm (red), or {lambda}excitation 495 nm, {lambda}emission 420–485 nm (green).

Polymerase Chain Reaction
Total cellular DNA was purified with a DNeasy Tissue KitTM using the supplier’s protocol (Qiagen; Valencia, CA; http://www.qiagen.com). DNA concentration and purity were checked spectrophotometrically at 260 nm and 280 nm, and by electrophoresis on 0.7% agarose minigels and ethidium bromide (EtBr) staining [14]. The sequences of the oligodeoxynucleotide primers used for PCR and RT-PCR (see below) were obtained from GenBank; specificity was checked by Basic Local Alignment Search Tool searches [15]. Primer design and annealing temperatures were chosen using VectorNTI v.7 (InforMax; Bethesda, MD; http://www.informaxinc.com). Primers were synthesized and purified by standard procedures (GenBase; La Jolla, CA). Nucleotide sequences were: mouse Sry (sex-determining region of the Y-chromosome; sense 5'-ATTTATGGTGTGGTCCCGTG; anti-sense 3'-GACACT TTAGCCCTCCGATG [underlined letters denote mouse primer mismatches with respect to the corresponding rat primers]), rat Pigr (polymeric immunoglobulin receptor gene, 5'-TTGGTTTCAAAGAAGCAGGAGGAG [the mouse 5'-strand contains a mismatch, a, at position 5]; 3'-GAGGTC CACATGAGTGACAGGAAG), rat INS1 (insulin gene, 5'-CATGGATGGCACTGGAGAAG; 3'-TCAGTAGCACA GCGTTCACC), mouse INS2 (5'-AGGAGCAGAGAGCA AGGGAC; 3'-CACCTGAAGGCCAGCAAGTC), rat COX1 (cytochrome C oxidase 1 gene, 5'-GCAGGAATAGTAGG GACAGCTTTG; 3'-GAGTAGAAATGATGGAGGAAG CAG), mouse COX1 (5'-GCAGAATTAGGTCAACCAG GTG; 3'-TGACACTCCAGCTAAATGAAGG), rat ß-actin (5'-CCCAGAGCAAGAGAGGTATC; 3'-GACCAGAGGC ATACAGGGAC), and jellyfish EGFP (5'-AGGACGACG GCAACTACAAG; 3'-GGGTGCTCAG GTAGTGGTTG).

Polymerase chain reaction was performed using a MiniCyclerTM (MJ Research; Boston, MA; http://www.mjr.com). Reaction mixtures (50 µl) contained 500 ng template DNA (unless noted), 25 pmol primer, 2 mM MgCl2, 0.2 mM nucleotide mix, and 2.5 units DNA Taq Polymerase (Promega). PCR conditions were: denaturation 5 minutes at 95°C, 40 cycles (30 seconds at 95°C, 30 seconds at 62°C, 30 seconds at 72°C), final extension 5 minutes at 72°C, hold at 4°C. Following amplification, the samples were analyzed immediately by gel electrophoresis or stored at -20°C. The amplified Pigr PCR products (10 µl) were electrophoresed on 3.5% agarose minigels or nondenaturing 5% polyacrylamide gels; other samples were analyzed on 1.5% agarose minigels. Gel bands were visualized by UV light and photographed with a Polaroid camera.

Pathogen screening was performed by the Research Animal Diagnostic Lab (University of Missouri-Columbia), which examined rat (Toolan’s rat parvovirus, Kilham’s rat virus, lymphocytic choriomeningitis virus, lactate dehydrogenase elevating virus, Mycoplasma sp., pneumonia virus of mice [PVM], rat cytomegalovirus, rat corona virus/sialodacryadenitis virus, rat parvovirus, and Sendai) and mouse (mouse hepatitis virus, minute virus of mice, mouse parvovirus, Mycoplasma sp., PVM, Sendai, and Theiler’s murine encephalomyelitis virus strain GDVII-mouse polio virus) pathogens. Additional para-pneumonia-like organisms (PPLO) screening was performed using primers and protocols provided in the American Type Culture Collection Mycoplasma Detection Kit v.2.0 [7]; 5 pg DNA of known standards were used as control templates.

Reverse Transcription Polymerase Chain Reaction   Total cellular RNA was purified using an RNeasyTM Minikit using the supplier’s protocol (Qiagen). RNA concentration and purity were checked spectrophotometrically at 260 nm and 280 nm, and by electrophoresis on 1.2% formaldehyde-agarose minigels and EtBr staining. RNA was treated with RQ1 RNase-free DNase (Promega) and stored at -70°C. Reverse transcription was performed with the ImProm-IITM Reverse Transcription SystemTM using reagents kits and protocols supplied by Promega. The primer sequences for rat were: ALB (sense 5'-GACTGCCCTGT GTGGAAGAC; anti-sense 3'-CGAAGTCACCCATCACC GTC), AFP (5'-AACAAGTATGGATTCTCAGG; 3'-ATT GATGCTCTCTTTGTCTG), cytochrome P450-1A2 (5'-GAACTACAAAGACAACGGTG; 3'-TCTTTCCACTGCT TCTCATC), and ß-actin (5'-CCCAGAGCAAGAGAGGC ATC; 3'-GACCAGAGGCATACAGGGAC); sequences for mouse were: ALB (5'-GACTGCCTTGTGTGGAGGAC; 3'-CAAAGTCATCCATGACAGTC), AFP (5'-AACAAG TATGGACTCTCAGG; 3'-ATGGATGCTCTCTTTGT CTG), cytochrome P450-1A2 (5'-GAACTACAAAGACA ATGGCG; 3'-TCTTTCCACTGCTTCTCATC), and ß-actin (5'-CCCAGAGCAAGAGAGGTATC; 3'-GACCAGAGGC ATACAGGGAC).

Polymerase chain reaction was performed with a prewarmed thermal cycler (94°C): 5 minutes at 95°C; 40 cycles for 30 seconds at 95°C; 30 seconds at annealing temperatures of 60°C for ß-actin, ALB, cytochrome P450-1A2, and 56°C for AFP; and 30 seconds at 72°C, final extension at 72°C for 5 minutes, hold at 4°C. Following amplification, the samples were stored at -20°C or analyzed immediately by electrophoresis on 1.5% agarose minigels. Reaction products on EtBr-stained gels were visualized and photographed as described above.

Digital Displays of Gel Electrophoresis Products
Gel photographs from separate experiments were scanned digitally. Image brightness was enhanced where noted using Paint Shop Pro (JASC Inc.; Minnetonka, MN; http://www.jasc.com). The files were recombined to generate figures and truncated to conserve space. Only the observed bands are shown; no spurious experimental bands were shielded from view.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rat LPC Lines Do Not Express Oval Cell-like Proteins or Inducible Cytochrome P450-1A2
To confirm expression of differentiated functions typical of oval cells and mature hepatocytes, we recharacterized LPC line 3(8)#21 and its derivative, 3(8)#21-EGFP (the line most studied). To eliminate complications of interpretation, purified 3(8)#21 P21 and 3(8)#21-EGFP P23 lines (cloned cells subjected to two consecutive 7-day cycles of growth off feeder layers) and STO cells were screened for PPLO; all were negative (Fig. 1AGo). Purified 3(8)#21-EGFP P23 cells were also negative for several infectious agents, including Sendai virus.



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  		Figure 1. Screening of LPCs and STO feeder layer cells for PPLO and hepatocyte-specific functions. A) PCR analyses for PPLO. Gel lanes: 1, 7 (100-bp ladder), 2 (M. pirum [323-bp], control), 3 (M. Laidlawii [219-bp, 426-bp], control), 4 (STO cells), 5 (purified 3(8)#21-EGFP P23), 6 (purified 3(8)#21 P21). B) RT-PCR analyses for rat ALB and rat AFP mRNA. Gel lanes: 1, 20 (100-bp ladder), 2–4 (neonatal rat liver, day 1), 5–7 (adult nursing rat liver), 8–10 (rat 7777 cells), 11–13 (purified 3(8)#21-EGFP P25, log-phase), 14–16 (purified 3(8)#21-EGFP P25, stationary-phase), 17–19 ({gamma}-irradiated STO cells, day 3). Each group of three gel lanes (left to right): ß-actin, ALB, AFP. C) RT-PCR analyses for rat cytochrome P450-1A2 mRNA. Gel lanes: 1, 14 (100-bp ladder), 2–3 (neonatal rat liver, day 1), 4–5 (adult nursing rat liver), 6–7 (rat 7777 cells), 8–9 (purified 3(8)#21-EGFP P25, log-phase), 10–11 (purified 3(8)#21-EGFP P25, stationary-phase), 12–13 ({gamma}-irradiated STO cells, day 3). Each group of two gel lanes (left to right): ß-actin, cytochrome P450-1A2. The experiments in each panel were repeated 3–5 times with identical results.

 
Prior observations indicate that both proliferation-competent LPC lines expressed ALB and AFP when cultured on {gamma}-irradiated STO cell feeder layers. In contrast, ALB and cytochrome P450-1A2 expression were maintained or induced, respectively [4]. Starting with log-phase 3(8)#21-EGFP P20 cells cultured on feeder layers, initial attempts to demonstrate AFP and ALB expression on Western blots revealed an ~68-kDa ALB-like band; however, no 70-kDa AFP-like band was detected (data not shown). Similar results were also obtained with purified 3(8)#21-EGFP P31 cells cultured off feeder layers. Paradoxically, however, Western blots revealed ALB-like bands in lysates from {gamma}-irradiated STO cells (cultured alone) in contrast to previous observations [4], and in 3(8)#21-EGFP cells pretreated 24 hours in vitro with 100 µg cycloheximide/ml, conditions that inhibit completely protein synthesis [16]. Positive ALB and AFP immunofluorescent signals were also detected in exhaustively washed fixed monolayers of purified 3(8)#21-EGFP P22 cells grown off feeder layers, and, paradoxically, in cultured 293 cells (which do not synthesize ALB or AFP). In both instances, control cultures stained with secondary Alexa Fluor 546 antibodies alone showed no detectable fluorescence. Nonetheless, the combined observations suggest that, by using the polyclonal immunochemical reagents currently available, immunochemical detection or serum protein uptake [17] artifacts might underlie the reported ALB and AFP expression in LPCs [4] and the discrepant observations associated with ALB expression in STO cells.

To circumvent these artifacts, rat ALB and rat AFP mRNA targets were investigated by single-round RT-PCR. Regardless of growth state, no expected 267-bp ALB or 321-bp AFP products were detected in purified 3(8)#21-EGFP P25 cells grown off feeder layers or on {gamma}-irradiated STO cells (Fig. 1BGo); in subsequent studies, similar results were observed using mouse-specific primers and control tissues (data not shown). Likewise, when purified 3(8)#21-EGFP cells grown on or off feeder layers were re-examined, no 390-bp cytochrome P450-1A2 mRNA product was detected using either rat primers (Fig. 1CGo) or control mouse primers and tissues (data not shown); {gamma}-irradiated STO cells were negative. It remains to be determined whether these three rat hepatocyte-specific mRNAs would have been detected by more sensitive assays, but their absence, as revealed by negative results of RT-PCR, support further the possibility of immunochemical artifacts.

Chromosomal and Mitochondrial DNAs from Rat LPC Lines Are of Mouse Origin
Given undetectable expression of ALB, AFP, and cytochrome P450-1A2 mRNAs, the possibility of contamination of rat LPC lines with STO feeder layer survivors was investigated at the suggestion of Dr. K.S. Koch. Since LPCs were female [4], STO cells were first examined for the Y-chromosome Sry element [18], with the hope that its presence would eliminate the contamination hypothesis. Control DNA templates from rat and mouse liver yielded PCR products of ~216 bp and ~580 bp, and ~216 bp, respectively, and no PCR product from mouse liver; no PCR products were observed from purified 3(8)#21-EGFP P23, 3(8)#21-EGFP P22, or 3(8)#21 P22 cells (Fig. 2AGo). Paradoxically, the control LPC ß-actin product (lane 5) migrated to positions observed for mouse tissue (lanes 3 and 4) and STO cell products (lane 8). The 580-bp rat Sry product might be due to nonspecific priming facilitated by mismatches between mouse primers and rat Sry sequence; size differences between rat and mouse ß-actin PCR products are as yet unexplained. The combined observations indicate that STO and LPC cells were and necessitated identification of species of origin.



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  		Figure 2. PCR analyses of gender and species of origin of STO cells and LPCs. A) Sry element. Mouse primers were designed to detect a 216-bp Sry product, expected for both rat and mouse DNA templates (top panel). Rat ß-actin primers were used to validate DNA quality (bottom panel); the reasons for the lower (~270-bp) than expected 731-bp control rat liver product (not shown), and the differences between the sizes of the observed rat and 240-bp mouse genomic products, are unclear. Gel lanes: 1, 9 (100-bp ladder), 2 ( rat), 3 ( mouse), 4 ( mouse), 5 (purified 3(8)#21-EGFP P23), 6 (purified 3(8)#21-EGFP P22), 7 (purified 3(8)#21 P22), 8 (STO cells). ND = not done. B) Pigr microsatellite. Primers were designed to detect 317-bp rat and 359-bp mouse products. The scanned gel photograph was digitally brightened to facilitate graphic visualization of the STO cell product. Gel lanes: 1, 8 (100-bp ladder), 2 (adult rat liver), 3 (STO cells), 4 (3(8)#21-EGFP P22), 5 (3(8)#21-EGFP P56), 6 (3(8)#21 P22), 7 (PC4 cells). C) Chromosomal, mitochondrial, and reporter genes. Panels of each gene and its expected product (top to bottom): Rat INS1 (353-bp), mouse INS2 (279-bp), rat COX1 (258-bp), mouse COX1 (354-bp), jellyfish EGFP (309-bp). Gel lanes: 1, 10 (100-bp ladder), 2 (mouse liver), 3 (3(8)#21-EGFP P22), 4 (3(8)#21-EGFP P56), 5 (3(8)#21 P22), 6 (3(8)#21-EGFP P23), 7 (PC4 cells), 8 (STO cells), 9 (rat liver). ND = not done. D) Rat and mouse COX1 [Set 1 UCSD vials]. The frozen vials were mailed directly to UCSD from AMC by Dr. L. Yin (shipping date = 9/00), and stored on arrival at -120°C. Cells in all vials were generated during the months of 7/00, or 8/00 (3(8)#21 P16) and 9/00 (clone 1(1)#6 P8). Gel lanes (where P = Pvial [passage number on vial]): 1, 26 (100-bp ladder), 2 (mouse liver), 3–4 (3(8)#21 P16), 5–6 (1(1) P5), 7–8 (1(1)#3 P10), 9–10 (1(1)#6 P8), 11–12 (1(3) P5), 13–14 (1(3)#1 P10), 15–16 (1(3)#3 P8), 17–18 (1(3)#4 P9), 19–20 (1(5) P5), 21–22 (2(7) P7), 23–24 (2(11) P6), 25 (rat liver). Each group of two gel lanes (left to right): growth on (+, where Pexperimental = Pvial + 1) or off (-, where Pexperimental = Pvial + 2) feeder layers. (E) Rat and mouse COX1 [Set 2 AMC vials]. The frozen vials were mailed directly to UCSD by Dr. Z. Ilic at AMC (shipping date = 8/02), and stored on arrival at -120°C. The dates of storage at AMC (at -120°C, unless noted) when the cells were generated are given in brackets. Gel lanes (where P = Pvial [vial number]): 1, 16 (100-bp ladder), 2 (mouse liver), 3 (1(1) P4 [3/00]), 4 (1(3) P4 [3/00]), 5 (1(5) P4 [3/00]), 6 (2(7) P2 [3/00]), 7 (2(11) P4 [3/00]), 8 (1(1)#6 P8 [9/00, at -80°C]), 9 (1(1)#6 P9 [9/00, at -80°C]), 10 (1(1)#3 P7 [8/99]), 11 (1(3)#1 P7 [8/99]), 12 (1(3)#3 P6 [4/00, at -80°C]), 13 (1(3)#4 P3 [8/99]), 14 (3(8)21-EGFP P12 [3/00]), 15 (rat liver). The experiments in each panel were repeated 3–5 times with identical results.

 
Therefore, DNA from authentic rat liver, STO cells, and several LPCs (3(8)#21-EGFP P22 and P56, 3(8)#21 P22, and PC4 [subcloned 3(8)#21-EGFP P28]) were analyzed for 3' untranslated region triplet repeat microsatellite polymorphisms that were encoded by rat [19] and mouse Pigr genes [20] located on chromosomes 13 and 1, respectively [15]. Except for rat liver, which generated an expected ~320- to 330-bp product, all other samples generated larger products of ~350- to 360-bp. Nondenaturing PAGE, performed to clarify these size differences, revealed an ~359-bp mouse-like product from STO and LPC lines; no major ~317-bp rat liver product was observed (Fig. 2BGo).

To avoid complications of interpretation caused by conformational polymorphisms in the Pigr microsatellite products (Fig. 2BGo, lane 2 [19, 21]), we searched for exon products from several unlinked cellular genes: chromosomal rat INS1 (353 bp) and mouse INS2 (279 bp) and maternally inherited mitochondrial rat and mouse COX1 (258 bp and 354 bp, respectively). The copy numbers of rat and mouse mitochondrial DNA sequences are ~4,000/cell [22, 23]. Again, only mouse products were detected in the cultured LPCs (Fig. 2CGo). Under the PCR conditions for mitochondrial COX1 targets, the presence of one cell equivalent would have been detected in the DNA templates, as indicated by DNA sample mixing and serial dilution control experiments (data not shown). The presence of the integrated retroviral vector encoded EGFP reporter gene was also investigated: the expected ~309-bp product was detected in 3(8)#21-EGFP P22, P23, P56, and PC4 cells, but neither in mouse and rat liver nor in STO and 3(8)#21 cells (Fig. 2CGo).

Metaphase Spreads Suggest Rat LPC Lines Are Derived from {gamma}-Irradiated STO Cells
Diploid rat (2N = 42) or mouse cells (2N = 40) are characterized by the predominance of metacentric or acrocentric chromosomes, respectively [24, 25]. To estimate ploidy and visualize centromere locations in the chromosomes of LPCs and unirradiated STO cells, GTG-banding was performed with purified 3(8)#21 (UCSD) P22 cells, 3(8)#21-EGFP [AMC] P23 cells, PC4 cells, and with STO cells. In each case, microscopic examination (1,000x) revealed that stained metaphase spreads contained ~60 chromosomes, the majority of which were acrocentric (K.S. Koch, personal communication of unpublished results).

Experimental Artifacts and Dedifferentiation Do Not Account for Loss of Function and Mouse DNA in Rat LPCs
To eliminate possibilities of artifacts of cell processing and vial mix-up errors at UCSD, including in vitro dedifferentiation of purified 3(8)#21-EGFP cells greater than P12, we characterized DNA from cells in two different sets of untampered frozen vials expected to contain >1.0–2.5 x 106 rat LPCs and <1.1–2.8 x 105 {gamma}-irradiated STO cells (~ratio, 9[rat]:1[mouse]): Set 1, DNA from the progeny of cells grown immediately on or off feeder layers at UCSD, and Set 2, DNA extracted directly from the cells in such vials. No rat COX1 products were found in either Set 1 (Fig. 2DGo) or Set 2 samples (Fig. 2EGo); only mouse COX1 products were observed. These results suggested that ≥99.98% of the cells in both sets of vials consisted of mouse-derived cells. This sensitivity is 500x greater than previously reported [26]; purified DNA and modified primer sequences may account for this improvement. DNA samples were examined using published rat and mouse COX1 primers, yet our conditions yielded PCR products of identical size with complete species specificity.

To investigate the possibility of selective recovery of cells from frozen vials containing mixtures of rat and mouse cells, DNA samples from fresh or from frozen (-70°C) and thawed (37°C) mixed populations of known rat and STO cells were analyzed for rat and mouse COX1 products. Rat 7777 cells were used as surrogates of the rat LPCs postulated to be present in the original vials. The results of a single experiment (Fig. 3Go) revealed that, using cell mixture ratios of 1[rat]:1[mouse], 9[rat]:1[mouse], and 1[rat]:9[mouse], the relative intensities of serially diluted rat and mouse PCR products were similar before and after freezing and at cell ratios of 1[rat]:1[mouse] and 1[rat]:9[mouse]. The sensitivities of detection of the 258-bp rat or 354-bp mouse products were equivalent (~10 pg) and independent of freezing and thawing (panels 4 and 8). At the ratio expected of the original vials (9[rat]:1[mouse]), detection of rat products was ~fivefold more sensitive than detection of mouse products.



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  		Figure 3. Detection of mouse and rat PCR products in mixtures of mouse STO and rat 7777 cells: effects of freezing and thawing. Fresh cells (top 4 panels) were harvested directly from cell cultures. Cells were frozen 8 days before thawing (bottom 4 panels). The numbers of cells in the different mixtures are given to the right of each panel. Gel lanes: 1, 16 (100-bp ladders); 2–8 and 9–15 (mouse or rat COX1, respectively). Control DNA template concentrations were 500 ng (lanes 2 and 15 [mouse liver tissue control], and lanes 8 and 9 [rat liver tissue control]). Experimental DNA template concentrations used for serial dilution studies (from top [panel 1] to bottom [panel 8]): first panel, lanes 3–7 (200, 100, 50, 20, and 10 pg) and 10–14 (200, 100, 50, 20, and 10 pg); second panel, lanes 3–7 (1,000, 500, 200, 100, and 50 pg) and 10–14 (200, 100, 50, 20, and 10 pg); third panel, lanes 3–7 (100, 50, 20, 10, and 5 pg) and 10–14 (2,000, 1,000, 500, 200, and 100 pg); fourth panel, lanes 3–7 (50, 20, 10, and 5 pg [STO only], and 500 ng [7777 only]) and 10–14 (50, 20, 10, and 5 pg [7777 only], and 500 ng [STO only]); fifth panel, lanes 3–7 (200, 100, 50, 20, and 10 pg) and 10–14 (200, 100, 50, 20, and 10 pg); sixth panel, lanes 3–7 (1,000, 500, 200, 100, and 50 pg) and 10–14 (200, 100, 50, 20, and 10 pg); seventh panel, lanes 3–7 (100, 50, 20, 10, and 5 pg) and 10–14 (1,000, 500, 200, 100, and 50 pg); eighth panel, lanes 3–7 (50, 20, 10, and 5 pg [STO only], and 500 ng [7777]) and 10–14 (50, 20, 10, and 5 pg [7777 only], and 500 ng [STO only]). Underlined numbers indicate estimated visual limits of detection.

 
High Levels of {gamma}-Radiation Kill STO Cells
STO cells double every 20 hours in standard media (data not shown). To validate the method of {gamma}-irradiation used to inhibit STO cell proliferation, STO cells were exposed to 5,300 rads and frozen as previously described [4]. Cells from thawed vials were cultured, and cell counts and viability were quantified through 28 days. Roughly 66% of plated cells survived thawing; ~17% of the inoculum survived 24 hours later (Fig. 4AGo). The attached survivors did not die immediately; instead, the cell number doubled during the next 48 hours (Fig. 4AGo), and, by day 3, healthy morphology was observed (Fig. 4BGo). Cell proliferation was not measurable beyond day 4, when cell deterioration and death began. By day 28, ~10% of the cells were attached and viable (relative to day 3), but marked morphological abnormalities (loss of epithelioid shape, pleiotropic nuclei, multiple nuclei/cell, flattened and enlarged cytoplasm, bizarre membrane processes) were apparent (Fig. 4CGo).



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  		Figure 4. Growth and survival of STO cells exposed to {gamma}-irradiation. Six million cells were plated into 10-cm plastic dishes. A) Growth and viability of cells cultured for 4 weeks. Cell counts were determined by standard procedures [16]. Viability was determined by trypan blue-exclusion. Triplicate dishes per time point were used for both sets of measurements, with counting errors ±10%. Growth curve (circles, solid line) and percentage of viable trypan-blue negative cells (triangles, dashed line). B) and C) Phase photomicrographs. Digital photomicrographs, three (panel B) and 28 days (panel C) postplating, were taken with a Nikon Diaphot microscope equipped with a Nikon Coolpix 990 digital camera. The inset bars give relative magnifications.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phenotypic and genotypic recharacterization of putative liver progenitor cell lines and strains reveals that previously described LPCs were derived from the {gamma}-irradiated STO feeder layers used to coculture cells from injured rat liver [4]. Despite tendencies of rat-mouse hybrids to retain mouse chromosomes preferentially [27], we consider that spontaneous LPC-STO cell fusion is unlikely to have accounted for the results described here, because we have detected no rat genes (Pigr, INS1, COX1) or gene products (ALB, AFP, cytochrome P450-1A2). Although these lines are Sendai virus-free, we cannot exclude the acquisition of unidentified syncytial viruses, and, therefore, cannot rigorously eliminate the possibility of rat-mouse chimerism.

Dedifferentiation of authentic rat LPC survivors is excluded since only mouse DNA sequences were detected. Alternatively, owing to putatively poorer viability or slower growth of the primary rat cells when grown in cocultures, it is possible that early passages retained rat cells while later passages lost them. This too is unlikely, since no rat DNA sequences were found in cells from LPC vials as early as P2. Moreover, all prior studies were performed with P5-P12 cells, yet examination of 3(8)#21 P12 cells, from which 3(8)#21-EGFP cells were derived, revealed only mouse DNA sequences. Therefore, prior characterization of 3(8)#21 and 3(8)#21-EGFP cells would appear artifactual. While we cannot be certain of the other lines and strains, artifact is likely, since, in all possible instances, LPCs from P2-P12 were assayed directly after being thawed and, again, only mouse DNA sequences were detected.

Differential cell fragility (rat > STO) might have caused selective recovery of viable mouse cells during phases of freezing and thawing. This possibility is unlikely from PCR results in a simulated experiment using mixtures of STO and rat cells; it could not apply to 3(8)#21-EGFP cells since their existence contradicts this possibility. However, LPCs were frequently frozen overnight at -80°C before storage at -120°C, or at -80°C for up to 2 weeks before use. The effects of storage at -80°C on selective recovery are unknown.

We suspect that overgrowth and selection for a fraction of growth-competent {gamma}-irradiated STO cells occurred during the early stages of construction of the lines (1 > P < 5) prior to, during, or after cloning, or during amplification prior to storage [4]. This interpretation is supported by null phenotypes (ALB-, AFP-, cytochrome P450-1A2- cells) of purified and cloned derivatives of 3(8)#21 and 3(8)#21-EGFP, and by identical genotypes (rat INS1-, mouse INS2+, rat COX1-, mouse COX1+) among subsets of 3(8)#21 and 3(8)#21-EGFP cells and all reported LPC lines and strains (rat COX1-, mouse COX1+) harvested from early passage vials, including purified and cloned LPCs ≥ P22 grown off feeder layers. This interpretation further suggests that, in the ancestral cocultures, retroviral vector pLNSX/SV/GFP transducedproliferation-competent {gamma}-irradiated STON+ cells instead of the intended rat LPC targets.

How might overgrowth and selection for {gamma}-irradiated STO or STON+ cells have happened? The answers are not readily apparent since log dose-response killing curves predict that overgrowth and selection should not have occurred. Theoretical fractions of survivors (S) exposed to a total dose of 5,300 rads should have ranged between S = 0.37(5300/60) ~7 x 10-39 to S = 0.37(5300/300) ~2 x 10-8, where 60 and 300 rads reflect the dose range of radiation required to kill mouse cells down to 37% survival [28]. Consequently, approximately one survivor would have been expected under the initially reported radiation (5.4 x 107 cells irradiated/tube) and plating conditions (1.5 x 106 {gamma}-irradiated cells/plate [4]). This suggests that only 1 of 34 dishes could have harbored one STO cell survivor. Clearly, theoretical predictions were contradicted even by the growth curves and trypan blue-exclusion studies described here, which indicated a small growth spurt early in culture and a small surviving fraction present 4 weeks later.

We suggest, therefore, that some distinctive property, such as abnormal karyotype or thioguanine-resistant ouabain-resistant properties of STO cells [5], conferred protection to {gamma}-radiation at AMC, particularly if a defective or miscalibrated machine delivered a lower than expected total radiation dose. For instance, an inadvertent low dose effect [29] could have significantly augmented the LD37 to 5,300 rads, increasing the numbers of proliferation-competent survivors. Lower AMC radiation dose rates were unlikely to have facilitated long-term survival; such effects usually occur at dose rates less than 100 rads/minute [28]. Alternatively, {gamma}-irradiated STO cell survivors might have suppressed the growth of cocultured rat LPCs through bystander effects conferred via paracrine secretions or cell-cell contacts [30]. Whether mutations occurred in single growth regulatory STO cell genes is unknown. On the one hand, mutations are improbable, since the predicted frequency of double strand breaks (DSBs)/gene should have been low. Assuming ~30,000 genes/genome [31] and ~40 DSBs/100 rads/cell, ~2,100 DSBs/genome (~0.07 DSBs/gene) are expected in STO cells exposed to 5,300 rads (estimates for human cells are ~19 DSBs/genome/Gy [32]). On the other hand, permanent growth alterations are suggested from differences in the population doubling times between unirradiated STO (~20 hours) and {gamma}-irradiated 3(8)#21-EGFP cells through P56 (~28 hours).

Since most previously reported LPC markers were not recharacterized, it remains possible that other markers including CD34, CD45, and Thy1.1 [4], might be associated with authentic STO cells or {gamma}-irradiated derivatives. In addition, the reported culture-dependent conversion of 3(8)#21-EGFP cells into bile-duct-like structures or into cells with hepatocyte-like morphology suggests that such cells might nevertheless harbor hepatic progenitor-cell-like properties. Until these possibilities are reinvestigated, however, all prior results must be judged inconclusive [4, 33–35]. Thus, the question of whether pluripotential hepatic LPCs are isolatable from allyl alcohol-treated rats remains unanswered.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are indebted to Dr. K.S. Koch for suggesting the hunt for the nature of LPCs and Pigr polymorphisms, for allowing us to examine and describe here her preliminary results of karyotyping studies of STO cells and rat LPCs, and for helpful discussion. We thank Dr. J. Ward (Department of Radiation Biology, UCSD) for helpful discussion; Dr. S. Gupta (Department of Medicine, Albert Einstein College of Medicine) for comments; Ms. K.H. Son and Mr. J. Aguilera for technical assistance; and Dr. S. Maeda for C57/B6-SvJ mouse tissue and mouse RNA. This work was supported in part by the National Institute of Environmental Health Sciences (5 P42 ES10337-02 [H.L.L.]) and the National Institutes of Health (DK57619[S.S.]).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Koch KS, Leffert HL. Normal liver progenitor cells in culture. In: Sell S, ed. Stem Cells Handbook. Totowa, NJ: Humana Press, 2003 (in press).

  2. Rogler LE. Selective bipotential differentiation of mouse embryonic hepatoblasts in vitro. Am J Pathol 1997;150:591–602.[Abstract]

  3. Kubota H, Reid LM. Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc Natl Acad Sci USA 2000;97:12132–12137.[Abstract/Free Full Text]

  4. Yin L, Sun M, Ilic Z et al. Derivation, characterization and phenotypic variation of hepatic progenitor cell lines isolated from adult rats. Hepatology 2002;35:315–324.[CrossRef][Medline]

  5. Ware LM, Axelrad AA. Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: evidence that congenic mouse strains SIM and SIM.R differ at the Fv-1 locus. Virology 1972;50:339–348.[CrossRef][Medline]

  6. Martin GR, Evans MJ. Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Natl Acad Sci USA 1975;72:1441–1445.[Abstract/Free Full Text]

  7. American Type Culture Collection. Available at: http://www.atcc.org. Accessed May 7, 2003.

  8. Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.[CrossRef][Medline]

  9. Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.[CrossRef][Medline]

  10. Aoki T, Koch KS, Leffert HL. Attenuation of gene expression by a trinucleotide repeat-rich tract from the terminal exon of the rat hepatic polymeric immunoglobulin gene. J Mol Biol 1997;267:229–236.[CrossRef][Medline]

  11. Verma RS, Babu A. Human Chromosomes, Manual of Basic Techniques. New York, NY: Pergammon Press, 1989:1–240.

  12. Lu XP, Koch KS, Lew DJ et al. Induction of cyclin mRNA and histone H1-kinase during liver regeneration. J Biol Chem 1992;267:2841–2844.[Abstract/Free Full Text]

  13. Sell S. Distribution of alpha-fetoprotein- and albumin-containing cells in the livers of Fischer rats fed four cycles of N-2-fluorenylacetamide. Cancer Res 1978;38:3107–3113.[Abstract/Free Full Text]

  14. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. In: Ford N, Nolan C, Ferguson M eds. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.

  15. GenBank. Available at http://www.ncbi.nlm.nih.gov. Accessed May 7, 2003.

  16. Koch KS, Leffert HL. Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 1979;18:153–163.[CrossRef][Medline]

  17. Rajagopal J, Anderson WJ, Kume S et al. Insulin staining of ES cell progeny from insulin uptake. Science 2003;299:363.[Free Full Text]

  18. Gubbay J, Collignon J, Koopman P et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990;346:245–250.[CrossRef][Medline]

  19. Koch KS, Gleiberman AG, Aoki T et al. Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene. Nucleic Acids Res 1995;23:1098–1112.[Abstract/Free Full Text]

  20. Kushiro A, Sato T. Polymeric immunoglobulin gene of mouse: sequence, structure and chromosomal location. Gene 1997;204:277–282.[CrossRef][Medline]

  21. Fabregat I, Koch KS, Aoki T et al. Functional pleiotropy of an intramolecular triplex-forming fragment from the 3' UTR of the rat Pigr gene. Physiol Genomics 2001;5:53–65.[Abstract/Free Full Text]

  22. Wiesner RJ, Ruegg JC, Morano I. Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues. Biochem Biophys Res Commun 1992;183:553–559.[CrossRef][Medline]

  23. Veltri KL, Espiritu M, Singh G. Distinct genomic copy number in mitochondria of different mammalian organs. J Cell Physiol 1990;143:160–164.[CrossRef][Medline]

  24. Yosida TH. Cytogenetics of the Black Rat. Tokyo: University of Tokyo Press, Baltimore: University Park Press, 1980:1–256.

  25. Silver LM. Mouse Genetics: Concepts and Applications. New York, NY: Oxford University Press, 1995:1–362.

  26. Parodi B, Aresu O, Bini D et al. Species identification and confirmation of human and animal cell lines: a PCR-based method. Biotechniques 2002;32:432–440.[Medline]

  27. Schneider JA, Weiss MC. Expression of differentiated function in hepatoma cell hybrids. I. Tyrosine aminotransferase in hepatoma-fibroblast hybrids. Proc Natl Acad Sci USA 1971;68:127–131.[Abstract/Free Full Text]

  28. Hall EJ. Radiobiology for the Radiologist, 2nd ed. Hagerstown, MD: Harper and Row, 1978:1–460.

  29. Skov KA. Radioresponsiveness at low doses: hyper-radiosensitivity and increased radioresistance in mammalian cells. Mutat Res 1999;430:241–253.[Medline]

  30. Mothersill C, Seymour C. Radiation-induced bystander effects: past history and future directions. Radiat Res 2001;155:759–767.[CrossRef][Medline]

  31. Lander ES, Linton LM, Birren B et al. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 2001;409:860–921.[CrossRef][Medline]

  32. Lobrich M, Rydberg B, Cooper P. Repair of x-ray induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends. Proc Natl Acad Sci USA 1995;92:12050–12054.[Abstract/Free Full Text]

  33. Yin L, Ilic Z, Titian D et al. Characterization of liver stem cell lines from allyl alcohol-injured rat liver. Amer Assoc Cancer Res 2001;42:354.

  34. Yin L, Sun M, Ilic Z et al. Plasticity of liver progenitor cell lines from allyl alcohol-injured rat liver. FASEB J 2001;LB267:49.

  35. Yin L, Arenas MS, Clark A et al. The potentiality of liver oval cells to differentiate into insulin-producing cells. Pancreatic Development, Proliferation and Stem Cells. National Institutes of Health (NIDDK, ADA, JDF), 2001:48a.

Received January 10, 2003; accepted for publication April 28, 2003.



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