A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90–98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10⁶ cells/T25 flask or 1 × 10⁵ cells/cm²). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.     

The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells

The PICM-19 pig liver stem cell line was cultured in space for nearly 16 d on the STS-126 mission to assess the effects of spaceflight on the liver’s parenchymal cells—PICM-19 cells to differentiate into either monolayers of fetal hepatocytes or 3-dimensional bile ductules (cholangiocytes). Semi-quantitative data included light microscopic assessments of final cell density, cell morphology, and response to glucagon stimulation and electron microscopic assessment of the cells’ ultrastructural features and cell-to-cell connections and physical relationships. Quantitative assessments included assays of hepatocyte detoxification functions, i.e., inducible P450 activities and urea production and quantitation of the mRNA levels of several liver-related genes. Three post-passage age groups were included: 4-d-, 10-d-, and 14-d-old cultures. In comparing flight vs. ground-control cultures 17 h after the space shuttle’s return to earth, no differences were found between the cultures with the exception being that some genes were differentially expressed. By light microscopy both young and older cultures, flight and ground, had grown and differentiated normally in the Opticell culture vessels. The PICM-19 cells had grown to approximately 75% confluency, had few signs of apoptosis or necrosis, and had either differentiated into monolayer patches of hepatocytes with biliary canaliculi visible between the cells or into 3-dimensional bile ductules with well-defined lumens. Ultrastructural features between flight and ground were similar with the PICM-19 cells displaying numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies, and occasional lipid vacuoles. Cell-to-cell arrangements were typical in both flight and ground-control samples; biliary canaliculi were well-formed between the PICM-19 cells, and the cells were sandwiched between the STO feeder cells. PICM-19 cells displayed inducible P450 activities. They produced urea in a glutamine-free medium and produced more urea in response to ammonia. The experiment’s aim to gather preliminary data on the PICM-19 cell line’s suitability as an in vitro model for assessments of liver function in microgravity was demonstrated, and differences between flight and ground-control cultures were minor.     

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

Summary Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4–8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7–10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.     

Isolation and characterization of a bovine trophectoderm cell line derived from a parthenogenetic blastocyst

A bovine trophectoderm cell line was established from a parthenogenetic in vitro-produced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first 10 days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 weeks. Thereafter, the cell culture was passaged at 1:10-1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n = 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measured by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.     

Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays

Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM-19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with β-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6β-OH-, 2α-OH- and 2β-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD₅₀ was calculated to be 14.9 ± 0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrene-treated cultures and untreated controls; CD₅₀ were 1.59 μM and 31 μM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.     

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Isolation and characterization of a bovine visceral endoderm cell line derived from a parthenogenetic blastocyst

Summary A cell line, BPE-1, was derived from a parthegogenetic 8-d in vitro-produced bovine blastocyst that produced a cell outgrowth on STO feeder cells. The BPE-1 cells resembled visceral endoderm previously cultured from blastocysts produced by in vitro fertilization (IVF). Analysis of the BPE-1 cells demonstrated that they produced serum proteins and were negative for interferon-tau production (a marker of trophectoderm). Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions in conjunction with desmosomes. Rough endoplasmic reticulum was prominent within the cells as were lipid vacuoles. Immunocytochemistry indicated the BPE-1 cells had robust microtubule networks. These cells have been growth for over 2 yr for multiple passages at 1∶10 or 1∶20 split ratios on STO feeder cells. The BPE-1 cell line presumably arose from embryonic cells that became diploid soon after parthenogenetic activation and development of the early embryo. However, metaphase spreads prepared at passage 41 indicated that the cell population had a hypodiploid (2n=60) unimodal chromosome content with a mode of 53 and a median and mean of 52. The cell line will be of interest for functional comparisons with bovine endoderm cell lines derived from IVF and nuclear transfer embryos. Disclaimer: Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Initial culture behaviour of rat blastocysts on selected feeder cell lines

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examinated the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3–6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat. © 1995 Wiley-Liss, Inc.     

New culture system for human embryonic stem cells: autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

Long-term culture of porcine bladder epithelial cells

Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured by plating with freshly irradiated LA7 feeder cells. In this way, bladder cells from five pigs were carried to confluency in passages 1, 4, 7, 7, and 13, amounting to at least 6, 18, 24, 26, and 45 doublings in culture, respectively, and none showed signs of slowed proliferation at the time of culture termination. Fibroblasts never became a prominent feature of these cultures, and their frequency was determined to be about 26 fibroblasts per 10(5) cells in passage 9. Pig bladder cells in 0.5% serum doubled in number in slightly over 3 d, whereas cells in 5.0% serum doubled in about 6 d. In fresh medium without feeder cells only minimal proliferation of bladder cells occurred. In LA7-conditioned medium the bladder cell numbers decreased, leading to the conclusion that the stimulus from LA7 cells is mechanically or physically transmitted. The bladder cells reacted with antibodies to keratins 7 and 18 but not to keratin 14 or vimentin. Tight junctions, visualized with an antibody to the ZO1 protein, connected all the cells to their neighbors. Most cells in passage 9 carried the diploid chromosome number of 38.     

A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig

Summary Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain. Alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum-free medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were inhibited by transforming growth factor β1 (TGFβ1) at concentrations ≥1 ng/ml. The cell cultures grew slowly with doubling times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [>100 population doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis.     

Culturing the epiblast cells of the pig blastocyst

Summary Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.     

Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

制备精原干细胞滋养层细胞条件的优化

目的:优化精原干细胞培养滋养层细胞的制备条件。方法:首先根据文献资料报道和实践经验确定丝裂霉素C作用STO细胞的浓度范围和时间范围,利用双因素优选法缩短丝裂霉素C处理STO细胞的试验范围。然后采用MTT检测法确定丝裂霉素C处理STO细胞的最佳浓度和时间。结果:滋养层细胞处理后不经过冷冻保存的情况下,17.64μg/ml 2 h处理条件的STO细胞在培养14天内细胞数量基本保持稳定,其它处理条件的细胞数量均有所增加;经冷冻保存的情况下,14.72μg/ml 2 h处理条件的STO细胞在培养14天内细胞数量基本保持稳定,其它处理条件的细胞数目均有所降低。结论:滋养层细胞处理后不经过冷冻保存的情况下,17.64μg/ml 2 h的处理条件是丝裂霉素C处理STO细胞的最佳条件;经冷冻保存的情况下,14.72μg/ml 2 h的处理条件是丝裂霉素C处理STO细胞的最佳条件。