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Perturbation of thyroid hormone (T 4 ) synthesis is known to cause numerous developmental, metabolic, and cognitive disorders in humans. Due to species differences in sensitivity to chemical exposures, there is a need for human-based in vitro approaches that recapitulate thyroid cellular architecture and T 4 production when screening. To address these limitations, primary human thyrocytes, isolated from healthy adult donor tissues and cryopreserved at passage one (p'1) were characterized for cellular composition, 3D follicular architecture, and thyroglobulin (TG)/T 4 expression and inhibition by prototype thyroid disrupting chemicals (TDC). Flow analysis of the post-thaw cell suspension showed >80% EpCAM-positive cells with 10%-50% CD90-positive cells. When seeded onto 96-well Matrigel ® -coated plates and treated with bovine thyroid stimulating hormone (TSH), thyrocytes formed 3D microtissues during the initial 4-5 days of culture. The microtissues exhibited a stable morphology and size over a 14-day culture period. TG and T 4 production were highest in microtissues when the proportion of CD90-positive cells, seeding density and thyroid stimulating hormone concentrations were between 10%-30%, 6K-12K cells per well, and 0.03-1 mIU/mL, respectively. At maximal TG and T 4 production levels, average microtissue diameters ranged between 50 and 200 µm. The T 4 IC 50 values for two prototype TPO inhibitors, 6-propyl-2-thiouracil and methimazole, were ∼0.7 µM and ∼0.5 µM, respectively, in microtissue cultures treated between days 9 and 14. Overall, p'1 cryopreserved primary human thyrocytes in 3D microtissue culture represent a promising new model system to prioritize potential TDC acting directly on the thyroid as part of a weight-of-evidence hazard characterization., Competing Interests: ER, EKB, T-NJ, SMA, JJO, DTP, KW, TS, PB, JC, SCP, JRW, and ELL were employed by LifeNet Health., (Copyright © 2024 Rogers, Breathwaite, Nguyen-Jones, Anderson, Odanga, Parks, Wolf, Stone, Balbuena, Chen, Presnell, Weaver and LeCluyse.)

Liver diseases, including NAFLD, are a growing worldwide health concern. Currently, there is a lack of suitable in vitro models that sustain basic primary human hepatocyte (PHH) morphology and functionality while supporting presentation of disease-associated phenotypic characteristics such as lipid accumulation and inflammasome activation. In TruVivo, an all-human triculture system (hTCS), basic metabolic functions were characterized in PHHs isolated from normal or diseased livers during two-weeks of culture. Decreases in albumin and urea levels and CYP3A4 activity were seen in diseased-origin PHHs compared to normal PHHs along with higher CYP2E1 expression. Positive expression of the macrophage markers CD68 and CD163 were seen in the diseased PHH preparations. Elevated levels of the pro-inflammatory cytokines IL-6 and MCP-1 and the fibrotic markers CK-18 and TGF-β were also measured. Gene expression of FASN, PCK1, and G6PC in the diseased PHHs was decreased compared to the normal PHHs. Further characterization revealed differences in lipogenesis and accumulation of intracellular lipids in normal and diseased PHHs when cultured with oleic acid and high glucose. TruVivo represents a promising new platform to study lipogenic mechanisms in normal and diseased populations due to the preservation of phenotypic differences over a prolonged culture period., (© 2024. The Author(s).)

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Purpose: This report explores the overlooked potential of bioprinting to automate biomanufacturing of simple tissue structures, such as the uniform deposition of (mono)layers of progenitor cells on sheetlike decellularized extracellular matrices (dECM). In this scenario, dECM serves as a biodegradable celldelivery matrix to provide enhanced regenerative microenvironments for tissue repair. The Tissue-Engineered Muscle Repair (TEMR) technology-where muscle progenitor cells are seeded onto a porcine bladder acellular matrix (BAM), serves as a representative testbed for bioprinting applications. Previous work demonstrated that TEMR implantation improved functional outcomes following VML injury in biologically relevant rodent models. Materials and Methods: In the described bioprinting system, a cell-laden hydrogel bioink is used to deposit high cell densities (1.4 × 105-3.5 × 105 cells/cm 2 ), onto both sides of the bladder acellular matrix as proof-of-concept. Results: These bioprinting methods achieve a reproducible and homogeneous distribution of cells, on both sides of the BAM scaffold, after just 24hrs, with cell viability as high as 98%. These preliminary results suggest bioprinting allows for improved dual-sided cell coverage compared to manual-seeding. Conclusions: Bioprinting can enable automated fabrication of TEMR constructs with high fidelity and scalability, while reducing biomanufacturing costs and timelines. Such bioprinting applications are underappreciated, yet critical, to expand the overall biomanufacturing paradigm for tissue engineered medical products. In addition, biofabrication of sheet-like implantable constructs, with cells deposited on both sides, is a process that is both scaffold and cell-type agnostic, and furthermore, is amenable to many geometries, and thus, additional tissue engineering applications beyond skeletal muscle.

The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents., Competing Interests: This work was supported by Organovo, Inc., San Diego. Deborah G. Nguyen and Sharon C. Presnell are employees of Organovo, Inc., San Diego. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

The human intestinal mucosa is a critical site for absorption, distribution, metabolism, and excretion (ADME)/Tox studies in drug development and is difficult to recapitulate in vitro. Using bioprinting, we generated three-dimensional (3D) intestinal tissue composed of human primary intestinal epithelial cells and myofibroblasts with architecture and function to model the native intestine. The 3D intestinal tissue demonstrates a polarized epithelium with tight junctions and specialized epithelial cell types and expresses functional and inducible CYP450 enzymes. The 3D intestinal tissues develop physiological barrier function, distinguish between high- and low-permeability compounds, and have functional P-gp and BCRP transporters. Biochemical and histological characterization demonstrate that 3D intestinal tissues can generate an injury response to compound-induced toxicity and inflammation. This model is compatible with existing preclinical assays and may be implemented as an additional bridge to clinical trials by enhancing safety and efficacy prediction in drug development., (Copyright © 2018 Organovo Inc. Published by Elsevier Inc. All rights reserved.)

Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

Compound-induced liver injury leading to fibrosis remains a challenge for the development of an Adverse Outcome Pathway useful for human risk assessment. Latency to detection and lack of early, systematically detectable biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Here, we demonstrate the utility of bioprinted tissue constructs comprising primary hepatocytes, hepatic stellate cells, and endothelial cells to model methotrexate- and thioacetamide-induced liver injury leading to fibrosis. Repeated, low-concentration exposure to these compounds enabled the detection and differentiation of multiple modes of liver injury, including hepatocellular damage, and progressive fibrogenesis characterized by the deposition and accumulation of fibrillar collagens in patterns analogous to those described in clinical samples obtained from patients with fibrotic liver injury. Transient cytokine production and upregulation of fibrosis-associated genes ACTA2 and COL1A1 mimics hallmark features of a classic wound-healing response. A surge in proinflammatory cytokines (eg, IL-8, IL-1β) during the early culture time period is followed by concentration- and treatment-dependent alterations in immunomodulatory and chemotactic cytokines such as IL-13, IL-6, and MCP-1. These combined data provide strong proof-of-concept that 3D bioprinted liver tissues can recapitulate drug-, chemical-, and TGF-β1-induced fibrogenesis at the cellular, molecular, and histological levels and underscore the value of the model for further exploration of compound-specific fibrogenic responses. This novel system will enable a more comprehensive characterization of key attributes unique to fibrogenic agents during the onset and progression of liver injury as well as mechanistic insights, thus improving compound risk assessment., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

Liver transplantation, either a partial liver from a living or deceased donor or a whole liver from a deceased donor, is the only curative therapy for severe end-stage liver disease. Only one-third of those on the liver transplant waiting list will be transplanted, and the demand for livers is projected to increase 23% in the next 20 years. Consequently, organ availability is an absolute constraint on the number of liver transplants that can be performed. Regenerative therapies aim to enhance liver tissue repair and regeneration by any means available (cell repopulation, tissue engineering, biomaterials, proteins, small molecules, and genes). Recent experimental work suggests that liver repopulation and engineered liver tissue are best suited to the task if an unlimited availability of functional induced pluripotent stem (iPS)-derived liver cells can be achieved. The derivation of iPS cells by reprogramming cell fate has opened up new lines of investigation, for instance, the generation of iPS-derived xenogeneic organs or the possibility of simply inducing the liver to reprogram its own hepatocyte function after injury. We reviewed current knowledge about liver repopulation, generation of engineered livers and reprogramming of liver function. We also discussed the numerous barriers that have to be overcome for clinical implementation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Myocardin (MYOCD) is a smooth and cardiac muscle-specific transcriptional coactivator that is required for the proper expression of contraction-related genes. Through its function to transactivate effector genes, MYOCD plays an essential role in mediating the switch between contractile and non-contractile phenotypes, particularly in smooth muscle cells (SMC). There are at least two known transcript variants of MYOCD that are expressed in SMC, differing only by the presence (+) or absence (Δ) of Exon 11. To date, no functional role has been assigned to the domain encoded by Exon 11, nor have any notable differences between the ability of each isoform to activate contraction-related genes been observed. In this study we compared sequences for Exon 11 among several mammalian species and identified a highly conserved, putative target sequence for glycogen synthase kinase 3 (GSK3) phosphorylation, suggesting a regulatory role for Exon 11 that can be modulated by alternative splicing. The function of Exon 11 was investigated by altering MYOCD splice selection in cultured porcine SMC with small interfering RNAs (siRNA) and specific chemical inhibitors, resulting in a relative increase in expression of ΔExon 11 variants in the endogenous pool of MYOCD mRNA. The relative increase in ΔExon 11 mRNAs correlated with a reduction of contractile phenotype in the porcine SMC as evidenced by morphological assessment and molecular analysis of effector genes. Together, these data suggest that MYOCD ΔExon 11 may participate in modulating SMC phenotype, potentially acting as a dominant-negative repressor of contraction-related genes., (Copyright © 2010 Wiley-Liss, Inc.)

Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.

Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4-7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding ("dO2 slope"). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized "dO2 slope" in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.

In recent years the concept of a stem cell has evolved to encompass the hypotheses that stem cells exist within many adult tissues, and that a common 'interchangeable' progenitor cell may exist within the bone marrow capable of regenerating and repairing tissues throughout the body. As more knowledge is gained about stem cells, their potential roles in disease processes, including the development and progression of cancer, have moved to the forefront. The underlying hypothesis of this review is that cell fate is determined by a combination of intrinsic and extrinsic factors; growth and differentiation are regulated through intracellular integration of a multitude of signals initiated by internal and external stimuli. The development of successful stem cell based therapies may depend on experimental approaches that consider both the intrinsic and extrinsic factors that control cell fate.

Human tissue xenograft models are currently the only tool for conducting in vivo analyses of intact human tissue. The goal of the present study was to develop reliable methods for successful generation of short-term primary tissue xenografts from benign and tumor-derived human prostate tissue. Primary human prostate xenografts were established in athymic nu/nu mice from eight of eight benign and five of five prostate cancer tissues, collected from a total of 10 patients who underwent radical prostatectomy for the treatment of prostate cancer. An average of 13 xenografts was established per specimen. Two tissue specimens were cryopreserved for >1 month before successful generation of prostate xenografts. After 1 month in vivo, xenograft tissues were harvested and examined regarding: gross evidence of vascularization; tissue morphology; proliferation; apoptosis; and expression of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins specific for basal cells in the prostate. Direct comparison of the original tissue specimen and the 1-month xenografts revealed similar histology; similar apoptotic and proliferative fractions in most cases; and comparable expression levels and expression patterns of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins. These data demonstrate that primary human prostate xenografts, benign and malignant, can be established routinely from human prostate tissue surgical specimens, and that the xenografts maintain tissue architecture and expression of key prostatic markers. The development of this methodology, including the technique for cryopreservation of human tissue, will allow multiple (successive) analyses of human prostate tissue to be conducted throughout time using a tissue sample derived from a single patient; and simultaneous analysis of human prostate tissues derived from a cohort of patients.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Human prostate cancer is initially dependent on androgens for growth, and androgen-dependent cells undergo apoptosis after castration. However, a subset of androgen-responsive cells survives and eventually proliferates in the absence of testicular androgen. The high levels of androgen receptor in both androgen-dependent and recurrent tumors led us to investigate androgen regulation of cell cycle proteins in human prostate cancer using the CWR22 xenograft. Cellular proliferation decreased dramatically in CWR22 tumors after castration. Testosterone propionate (TP) treatment of castrated mice restored cellular proliferation after 24-48 hours. Growth of CWR22 tumors in the absence of testicular androgen recurred several months after castration. CDK1 and CDK2, and cyclin A and cyclin B1 messenger RNAs were decreased 6 days after castration, increased 6-12 hours after TP treatment, and were expressed at high levels in recurrent CWR22 tumors. Coimmunoprecipitated cyclin B1/CDK1 and cyclin D1/CDK4 protein complexes decreased after castration and increased after TP treatment of castrated mice. In addition, CDK1 and CDK2 kinase activities were upregulated by androgen in parallel with hyperphosphorylation of retinoblastoma (Rb) protein. Despite the absence of testicular androgen in recurrent CWR22, the levels of these androgen-regulated cyclin/ CDK protein complexes and hyperphosphorylation of Rb were equal to or greater than in tumors from intact mice. The results indicate that androgen receptor regulates cellular proliferation by control of CDK and cyclins at the transcriptional level and by post-translational modifications that influence cell cycle protein activity.

Normal prostate epithelial cells are difficult to propagate in vitro without experimental immortalization. The goal of this study was to isolate and characterize a propagable epithelial cell line from normal adult rat prostate. Enrichment of proliferation-competent cells was accomplished in vivo by initiating a single cycle of prostatic involution/regeneration. The RPE-F344 cell line was established from an androgen-deprived, involuted prostate four days after the initiation of regeneration by administration of testosterone. The cell line has been cultured in vitro for >50 passages, forms a uniform monolayer in culture, exhibits contact inhibition at confluence, and does not form colonies in soft agar. Immunocytochemical and RT-PCR analyses demonstrated that the RPE-F344 cells express anti-apoptotic genes associated with cell survival, and several growth factor receptors important in prostate development and homeostasis. RPE-F344 cells are p27kip1 negative, telomerase positive, and express high molecular weight cytokeratins specific for prostatic basal cells. They also express low levels of androgen receptor (AR) and prostatic acid phosphatase (PAP); features associated with secretory luminal epithelial cells. RPE-F344 cells are maintained in vitro without androgen supplementation, but addition of 15nM dihydrotesterone (DHT) to the culture media results in a significant but transient enhancement of cellular proliferation. Establishment of RPE-F344-like colonies from rat prostate is limited to the ventral and dorsal lobes of the prostate 2-4 days after initiation of regeneration, suggesting that RPE-F344 cells may originate from a stem cell-like compartment that is responsible for regenerative repopulation.

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of some rat liver tumor cell lines. In the present study, possible molecular mechanisms of human 11p11.2-p12-mediated liver tumor suppression were investigated by examining gene expression patterns in suppressed and non-suppressed microcell hybrid (MCH) cell lines. The parental rat liver tumor cell lines (GN6TF and GP7TB) express moderate levels of p53 mRNA and protein, overexpress mRNAs for c-H-ras, c-myc, and TGFá, and do not express detectable levels of WT1 mRNA or protein. Suppression of tumorigenicity by human chromosome 11p11.2-p12 was not accompanied by significant alterations in the levels of expression of p53, c-myc, or TGFá. Expression of c-H-ras was decreased significantly in both suppressed and non-suppressed MCH cell lines, suggesting that down-regulation of c-H-ras is not directly responsible for tumor suppression. In contrast, the level of expression of WT1 correlated precisely with tumor suppression in this model system. All suppressed MCH cell lines expressed WT1 mRNA and protein at levels comparable to that of untransformed rat liver epithelial cells (WB-F344), whereas only trace WT1 mRNA and protein were detected in a non-suppressed MCH cell line. PCR analysis demonstrated that two suppressed MCH cell lines do not carry the human WT1 gene, indicating that WT1 expression in these lines originates from the rat locus. Furthermore, RT-PCR analysis showed that each of the four known splice variants of the WT1 mRNA are expressed in these suppressed MCH cell lines, recapitulating the expression pattern observed in the untransformed rat liver epithelial cells. Re-expression of tumorigenicity by suppressed MCH cell lines was accompanied by the coordinate loss of human chromosome 11p11.2-p12 and of WT1 gene expression, suggesting that one or more human 11p11.2-p12 genes are required for sustained expression of WT1 in these cell lines. Together, these results suggest that the molecular mechanism governing human chromosome 11p11.2-p12-mediated liver tumor suppression may involve induction of rat WT1 gene expression under the direct or indirect transcriptional regulation of a genetic locus (or loci) on human 11p11.2-p12.

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.

Recently, a novel strategy for nucleotide exchange of target DNA using chimeric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be transfected into cells, remain stable within the cells, and migrate to the nucleus. We have in this study used 42 similar constructs for targeting six different human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoids, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts and hepatocytes. However, no nucleotide exchange was detected in any of the targeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during the first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). Furthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. However, cloning of the PCR products revealed no nucleotide exchange. In our view, the most likely explanation is that the initial false positive result originates from a PCR artifact created by the CO itself. Our results imply that an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.

A model of spontaneous malignant transformation was used to evaluate the molecular changes that take place in WB-F344 rat liver epithelial cells during neoplastic transformation and tumorigenesis. A comparison of wild-type low-passage WB-F344 cells to spontaneously transformed tumor cell lines revealed that the majority of the tumor cell lines have an increased capacity for autonomous proliferation and motility when maintained in serum-free media. In the current study, we show that c-met is expressed at some level in wild-type WB-F344 cells and in all of the spontaneously transformed tumor cell lines, and that 9/16 of the tumor cell lines have acquired hepatocyte growth factor (HGF) expression. In vitro growth of HGF-expressing tumor cell lines is inhibited as much as 68% by the addition of neutralizing antibodies to HGF or antisense HGF oligonucleotides, indicating that the production of HGF by the tumor cells is partially responsible for driving autonomous proliferation in a subset of tumor cell lines. Furthermore, conditioned media collected from HGF-expressing tumor cell lines stimulates DNA synthesis in wild-type WB-F344 cells, and this effect can be abrogated by pre-incubation of the conditioned media with neutralizing antibodies to HGF. Because HGF is a motility-promoting growth factor, all cell lines were evaluated to determine if expression of HGF stimulated motogenesis. All tumor cell lines (regardless of HGF expression) were highly motile in comparison with wild-type WB-F344 cells, with a 3.5-fold to 20-fold greater number of motile cells. The high basal rate of motility characteristic of the tumor cell lines is not a result of the production of HGF, because it is also a property of the cell lines that do not express HGF messenger RNA. Furthermore, tumor cell motility is not inhibited by antisense oligonucleotides or neutralizing antibodies. Establishment of an autocrine HGF/c-met loop in a subset of spontaneously transformed WB-F344 cell lines may influence development and/or expression of the tumorigenic phenotype by driving cellular proliferation.

The genomic evolution of a cohort of WB-F344 rat liver epithelial cell lineages undergoing spontaneous neoplastic transformation was followed to define the mechanistic relationship between genomic instability and progression to the neoplastic phenotype. Eighteen independent populations of WB-F344 cells (initiated from a single diploid-founding population) were subjected to 12 cycles of selective growth at confluent cell density, and cellular DNA contents were measured after each selection cycle. Flow cytometry demonstrated significant gains in the amount of G1 DNA after selection cycles 3, 6, and 7 in 44% (8 of 18), 89% (16 of 18), and 39% (7 of 18) of the cell populations, respectively. All populations subsequently lost DNA and returned to a diploid or pseudo-diploid DNA content within 1 to 2 selection cycles after the appearance of an increased DNA content. Additionally, appearance and subsequent disappearance of aneuploid or tetraploid subpopulations was observed in 11% (2 of 18) and 83% (15 of 18) of the experimental lineages, respectively. Although perturbations of G1 DNA content were apparent as early as selection cycle 3, at least 8 cycles of selective growth were required for the acquisition of tumorigenicity. While the independent lineages demonstrated significant fluctuations in G1 DNA content between selection cycles 3 and 8, the majority (11 of 13) of the populations contained a diploid or pseudo-diploid DNA content at the time tumorigenicity was expressed. Genomic instability preceded the acquisition of tumorigenic potential in rat liver epithelial cells subjected to selective growth conditions of maintenance at confluence, and may be required for its expression.

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA repeats onto the ends of chromosomes. More than 85% of human cancers express telomerase activity and a large proportion of human hepatocellular carcinomas are positive. To study the role of telomerase expression in rat hepatocarcinogenesis, telomerase activity was assayed in various rat tissues and in two types of liver epithelial cells: hepatocytes and hepatic epithelial stem-like cells. In the present study, we demonstrate that telomerase activity in rats is tissue-specific and stable with animal aging. Liver and testis were found to be telomerase positive, spleen had low or no activity, and kidney was negative. Telomerase activity did not change significantly in 18 month-old rats compared to 2 month-old rats, but was moderately (twofold) increased during liver regeneration induced by a 2/3's partial hepatectomy. Telomerase activity was detected in isolated rat hepatocytes and low passage hepatic epithelial stem-like cells (WB-F344). Telomerase activity displayed significant variations in a propagable clone of WB-F344 cells. At low passage levels after establishment in vitro (passages 4-9) non-tumorigenic WB-F344 cells expressed telomerase activity. During further in vitro passaging these cells lost expression of telomerase. Expression of telomerase in the tumor-derived lines of WB-F344 cells but not in the selectively cycled, parental lineages of these cells suggests that there may be a role for telomerase in hepatocarcinogenesis.

We have previously shown that rat liver epithelial cells (RLEC) transfected with and constitutively expressing transforming growth factor-alpha (TGF-alpha) have an enhanced mitogenic response to hepatocyte growth factor (HGF). In the study reported here, we examined tumor clones derived from the TGF-alpha transfectants with respect to mitogenic response to HGF. Tumor cell lines that expressed TGF-alpha responded to HGF with a greater increase in DNA synthesis than did the nontransfected parental RLEC (pRLEC). The tumor clones had also acquired a lower threshold for HGF response, which enabled them to undergo significant DNA synthesis at a low concentration of HGF that did not evoke a response in the pRLEC or TGF-alpha transfectants. We investigated the mechanisms by which TGF-alpha expression may influence the HGF/c-met pathway. We showed that most TGF-alpha transfectants and tumor cells displayed increases in c-met mRNA and protein, indicating that the enhanced HGF response may be due in part to an increase in the amount of receptor present. However, in all transfectants and tumor clones that constitutively expressed TGF-alpha, c-met was tyrosine phosphorylated in the absence of ligand (HGF) or other exogenous growth factors. These data suggest that induction of c-met mRNA and transactivation of c-met may be a sequela of the constitutive expression of TGF-alpha and that constitutive activation of the epidermal growth factor receptor pathway leads to phosphorylation and activation of c-met. These studies provide evidence for a novel mechanism of communication between epidermal growth factor receptor and c-met pathways that may partially explain the synergistic effects reported between TGF-alpha and HGF.

Overexpression of both transforming growth factor (TGF)-alpha and c-myc is consistently reported in hepatic tumors. We transfected rat liver epithelial cells (RLECs) with expression vectors for TGF-alpha, c-myc, or both and analyzed the morphology, biological properties, and tumorigenicity of clones that overexpressed these genes. The transfectants were morphologically indistinguishable from the parental RLECs, but the overexpression of TGF-alpha resulted in changes in growth properties and an enhanced response to the mitogenic effects of hepatocyte growth factor. The concomitant overexpression of c-myc decreased growth factor requirements of the TGF-alpha lc-myc clones compared with RLEC and TGF-alpha clones. The TGF-alpha and TGF-alpha lc-myc clones were tumorigenic in nude mice at frequencies of 27% and 53%, respectively, indicating that the genes cooperate in malignant transformation. However, the untransformed nature and low tumorigenicity of the transfectants suggest that transformation depends on other cellular events in addition to the overexpression of TGF-alpha or c-myc. Characterization of tumor cell lines showed that in contrast to the transfectants, the tumor clones were morphologically transformed, capable of autonomous growth and anchorage-independent growth, and aggressively tumorigenic with a frequency of 100%. Clearly, the tumor cells differed from the transfectants and had undergone biological or genetic alterations (or both) as a consequence of the overexpression of TGF-alpha or c-myc. Our data suggest that the overexpression of TGF-alpha leads to enhanced responsiveness to hepatocyte growth factor, whereas the concomitant overexpression of c-myc confers growth-factor independence, providing a potential explanation of the mechanisms by which the overexpression of these genes results in transformation.