Targeting Epigenetic Regulators with HDAC and BET Inhibitors to Modulate Muscle Wasting.

Epigenetic changes contribute to the profound alteration in the transcriptional program associated with the onset and progression of muscle wasting in several pathological conditions. Although HDACs and their inhibitors have been extensively studied in the field of muscular dystrophies, the potential of epigenetic inhibitors has only been marginally explored in other disorders associated with muscle atrophy, such as in cancer cachexia and sarcopenia. BET inhibitors represent a novel class of recently developed epigenetic drugs that display beneficial effects in a variety of diseases beyond malignancies. Based on the preliminary in vitro and preclinical data, HDACs and BET proteins contribute to the pathogenesis of cancer cachexia and sarcopenia, modulating processes related to skeletal muscle mass maintenance and/or metabolism. Thus, epigenetic drugs targeting HDACs and BET proteins may emerge as promising strategies to reverse the catabolic phenotype associated with cachexia and sarcopenia. Further preclinical studies are warranted to delve deeper into the molecular mechanisms associated with the functions of HDACs and BET proteins in muscle atrophy and to establish whether their epigenetic inhibitors represent a prospective therapeutic avenue to alleviate muscle wasting.

DCLK1, a Putative Stem Cell Marker in Human Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). APPROACH AND RESULTS: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine-rich repeat-containing G protein-coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real-time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real-time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme-linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared with unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in a dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls. CONCLUSIONS: DCLK1 characterizes a specific CSC subpopulation of iCCACD133+ and pCCALGR5+ , and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.

Exercise and Exercise Mimetics for the Treatment of Musculoskeletal Disorders.

PURPOSE OF REVIEW: The incidence of musculoskeletal disorders affecting bones, joints, and muscles is dramatically increasing in parallel with the increased longevity of the worldwide population, severely impacting on the individual's quality of life and on the healthcare costs. Inactivity and sedentary lifestyle are nowadays considered the main drivers of age-associated musculoskeletal disorders and exercise may counteract such alterations also in other bone- and muscle-centered disorders. This review aims at clarifying the potential use of exercise training to improve musculoskeletal health. RECENT FINDINGS: Both the skeletal muscle and the bone are involved in a complex crosstalk determining, in part through tissue-specific and inflammatory/immune released factors, the occurrence of musculoskeletal disorders. Exercise is able to modulate the levels of those molecules and several associated molecular pathways. Evidence from preclinical and clinical trials supports the adoption of exercise and the future use of exercise mimicking drugs will optimize the care of individuals with musculoskeletal disorders.

SMYD3 promotes the epithelial-mesenchymal transition in breast cancer.

SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGFß-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGFß-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGFß-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.

The methyltransferase SMYD3 mediates the recruitment of transcriptional cofactors at the myostatin and c-Met genes and regulates skeletal muscle atrophy.

Elucidating the epigenetic mechanisms underlying muscle mass determination and skeletal muscle wasting holds the potential of identifying molecular pathways that constitute possible drug targets. Here, we report that the methyltransferase SMYD3 modulates myostatin and c-Met transcription in primary skeletal muscle cells and C2C12 myogenic cells. SMYD3 targets the myostatin and c-Met genes and participates in the recruitment of the bromodomain protein BRD4 to their regulatory regions through protein-protein interaction. By recruiting BRD4, SMYD3 favors chromatin engagement of the pause-release factor p-TEFb (positive transcription elongation factor) and elongation of Ser2-phosphorylated RNA polymerase II (PolIISer2P). Reducing SMYD3 decreases myostatin and c-Met transcription, thus protecting from glucocorticoid-induced myotube atrophy. Supporting functional relevance of the SMYD3/BRD4 interaction, BRD4 pharmacological blockade by the small molecule JQ1 prevents dexamethasone-induced myostatin and atrogene up-regulation and spares myotube atrophy. Importantly, in a mouse model of dexamethasone-induced skeletal muscle atrophy, SMYD3 depletion prevents muscle loss and fiber size decrease. These findings reveal a mechanistic link between SMYD3/BRD4-dependent transcriptional regulation, muscle mass determination, and skeletal muscle atrophy and further encourage testing of small molecules targeting specific epigenetic regulators in animal models of muscle wasting.

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins by JQ1 Unravels a Novel Epigenetic Modulation to Control Lipid Homeostasis.

The homeostatic control of lipid metabolism is essential for many fundamental physiological processes. A deep understanding of its regulatory mechanisms is pivotal to unravel prospective physiopathological factors and to identify novel molecular targets that could be employed to design promising therapies in the management of lipid disorders. Here, we investigated the role of bromodomain and extraterminal domain (BET) proteins in the regulation of lipid metabolism. To reach this aim, we used a loss-of-function approach by treating HepG2 cells with JQ1, a powerful and selective BET inhibitor. The main results demonstrated that BET inhibition by JQ1 efficiently decreases intracellular lipid content, determining a significant modulation of proteins involved in lipid biosynthesis, uptake and intracellular trafficking. Importantly, the capability of BET inhibition to slow down cell proliferation is dependent on the modulation of cholesterol metabolism. Taken together, these data highlight a novel epigenetic mechanism involved in the regulation of lipid homeostasis.

A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth.

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.

The BET inhibitor JQ1 targets fat metabolism and counteracts obesity.

INTRODUCTION: Obesity, one of the most frequent health problems in the adult population, is a condition characterized by excessive white adipose tissue accumulation and accompanied by the increased risk to develop other disorders such as type II diabetes, cardiovascular disorders, physical disability, frailty and sarcopenia. Total fat mass frequently increases during aging, often coexisting with sarcopenia, thus resulting in an emerging condition defined sarcopenic obesity (SO). Our previous data demonstrated the relevant role of the bromo and extra-terminal domain (BET) proteins inhibitor JQ1 in attenuating inflammation and fibrosis in sarcopenic mice. Moreover, we preliminarily observed that JQ1 administration markedly reduces white adipose tissue mass, suggesting a potential role of BET proteins on visceral fat deposition during aging. OBJECTIVES: Starting from those observations, the aim of this study was to investigate the ability of JQ1 to reduce adiposity in a chronic diet-induced obesity (DIO) mouse model mimicking the human metabolic syndrome. METHODS: Male C57BL/6J mice were divided in subgroups, either fed a standard diet or a high fat diet for 22 or 12 weeks, treated over the last 14 days with JQ1 or with vehicle. RESULTS: The results showed that JQ1 administration reduces fat mass, preserving skeletal muscle mass and function. A direct JQ1 lipolytic effect was demonstrated on mature adipocyte cultures. JQ1-mediated loss of adipose tissue mass was not associated with systemic inflammation or with lipid accumulation in muscle and liver. JQ1 administration did not impinge on skeletal muscle metabolism and oxidative capability, as shown by the lack of significant impact on mitochondrial mass and biogenesis. CONCLUSION: In conclusion, the current data highlight a potential benefit of JQ1 administration to counteract obesity, suggesting epigenetic modulation as a prospective target in the treatment of obesity and sarcopenic obesity, despite the underlying multiorgan molecular mechanism is still not completely elucidated.

Tackling skeletal muscle cells epigenome in the next-generation sequencing era.

Recent advances in high-throughput technologies have transformed methodologies employed to study cell-specific epigenomes and the approaches to investigate complex cellular phenotypes. Application of next-generation sequencing technology in the skeletal muscle differentiation field is rapidly extending our knowledge on how chromatin modifications, transcription factors and chromatin regulators orchestrate gene expression pathways guiding myogenesis. Here, we review recent biological insights gained by the application of next-generation sequencing techniques to decode the epigenetic profile and gene regulatory networks underlying skeletal muscle differentiation.

Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3.

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 µM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.

MyoD regulates skeletal myogenesis. Since proteins associated with MyoD exert regulatory functions, their identification is expected to contribute important insights into the mechanisms governing gene expression in skeletal muscle. We have found that the RNA helicases p68/p72 are MyoD-associated proteins and that the noncoding RNA SRA also immunoprecipitates with MyoD. In vitro and in vivo experiments indicated that both p68/p72 and SRA are coactivators of MyoD. RNA interference toward either p68/p72 or SRA prevented proper activation of muscle gene expression and cell differentiation. Unexpectedly, reducing the levels of p68/p72 proteins impaired recruitment of the TATA binding protein TBP; RNA polymerase II; and the catalytic subunit of the ATPase SWI/SNF complex, Brg-1, and hindered chromatin remodeling. These findings reveal that p68/p72 play a critical role in promoting the assembly of proteins required for the formation of the transcription initiation complex and chromatin remodeling.

In vitro profiling of epigenetic modifications underlying heavy metal toxicity of tungsten-alloy and its components.

Tungsten-alloy has carcinogenic potential as demonstrated by cancer development in rats with intramuscular implanted tungsten-alloy pellets. This suggests a potential involvement of epigenetic events previously implicated as environmental triggers of cancer. Here, we tested metal induced cytotoxicity and epigenetic modifications including H3 acetylation, H3-Ser10 phosphorylation and H3-K4 trimethylation. We exposed human embryonic kidney (HEK293), human neuroepithelioma (SKNMC), and mouse myoblast (C2C12) cultures for 1-day and hippocampal primary neuronal cultures for 1-week to 50-200 µg/ml of tungsten-alloy (91% tungsten/6% nickel/3% cobalt), tungsten, nickel, and cobalt. We also examined the potential role of intracellular calcium in metal mediated histone modifications by addition of calcium channel blockers/chelators to the metal solutions. Tungsten and its alloy showed cytotoxicity at concentrations > 50 µg/ml, while we found significant toxicity with cobalt and nickel for most tested concentrations. Diverse cell-specific toxic effects were observed, with C2C12 being relatively resistant to tungsten-alloy mediated toxic impact. Tungsten-alloy, but not tungsten, caused almost complete dephosphorylation of H3-Ser10 in C2C12 and hippocampal primary neuronal cultures with H3-hypoacetylation in C2C12. Dramatic H3-Ser10 dephosphorylation was found in all cobalt treated cultures with a decrease in H3 pan-acetylation in C2C12, SKNMC and HEK293. Trimethylation of H3-K4 was not affected. Both tungsten-alloy and cobalt mediated H3-Ser10 dephosphorylation were reversed with BAPTA-AM, highlighting the role of intracellular calcium, confirmed with 2-photon calcium imaging. In summary, our results for the first time reveal epigenetic modifications triggered by tungsten-alloy exposure in C2C12 and hippocampal primary neuronal cultures suggesting the underlying synergistic effects of tungsten, nickel and cobalt mediated by changes in intracellular calcium homeostasis and buffering.

The Lysine Methylase SMYD3 Modulates Mesendodermal Commitment during Development.

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

Sarcopenia Diagnosis: Reliability of the Ultrasound Assessment of the Tibialis Anterior Muscle as an Alternative Evaluation Tool.

Sarcopenia is a skeletal muscle disorder characterized by reduced muscle mass, strength, and performance. Muscle ultrasound can be helpful in assessing muscle mass, quality, and architecture, and thus possibly useful for diagnosing or screening sarcopenia. The objective of this study was to evaluate the reliability of ultrasound assessment of tibialis anterior muscle in sarcopenia diagnosis. We included subjects undergoing total or partial hip replacement, comparing measures with a healthy control group. We measured the following parameters: tibialis anterior muscle thickness, echogenicity, architecture, stiffness, skeletal muscle index (SMI), hand grip strength, and sarcopenia related quality of life evaluated through the SarQoL questionnaire. We included 33 participants with a mean age of 54.97 ± 23.91 years. In the study group we found reduced tibialis anterior muscle thickness compared to the healthy control group (19.49 ± 4.92 vs. 28.94 ± 3.63 mm, p < 0.05) with significant correlation with SarQoL values (r = 0.80, p < 0.05), dynamometer hand strength (r = 0.72, p < 0.05) and SMI (r = 0.76, p < 0.05). Moreover, we found reduced stiffness (32.21 ± 12.31 vs. 27.07 ± 8.04 Kpa, p < 0.05). AUC measures of ROC curves were 0.89 predicting reduced muscle strength, and 0.97 predicting reduced SMI for tibialis anterior muscle thickness, while they were 0.73 and 0.85, respectively, for muscle stiffness. Our findings showed that ultrasound assessment of tibialis anterior muscle might be considered a reliable measurement tool to evaluate sarcopenia.

SMYD3: An Oncogenic Driver Targeting Epigenetic Regulation and Signaling Pathways.

SMYD3 is a member of the SMYD lysine methylase family and plays an important role in the methylation of various histone and non-histone targets. Aberrant SMYD3 expression contributes to carcinogenesis and SMYD3 upregulation was proposed as a prognostic marker in various solid cancers. Here we summarize SMYD3-mediated regulatory mechanisms, which are implicated in the pathophysiology of cancer, as drivers of distinct oncogenic pathways. We describe SMYD3-dependent mechanisms affecting cancer progression, highlighting SMYD3 interplay with proteins and RNAs involved in the regulation of cancer cell proliferation, migration and invasion. We also address the effectiveness and mechanisms of action for the currently available SMYD3 inhibitors. The findings analyzed herein demonstrate that a complex network of SMYD3-mediated cytoplasmic and nuclear interactions promote oncogenesis across different cancer types. These evidences depict SMYD3 as a modulator of the transcriptional response and of key signaling pathways, orchestrating multiple oncogenic inputs and ultimately, promoting transcriptional reprogramming and tumor transformation. Further insights into the oncogenic role of SMYD3 and its targeting of different synergistic oncogenic signals may be beneficial for effective cancer treatment.

BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

Targeting SMYD3 to Sensitize Homologous Recombination-Proficient Tumors to PARP-Mediated Synthetic Lethality.

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

Deacetylase inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin.

Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.

Mechanisms underlying the transcriptional regulation of skeletal myogenesis.

During skeletal myogenesis, chromatin-modifying enzymes are engaged at discrete genomic regions by transcription factors that recognize sequence-specific DNA motifs located at muscle gene regulatory regions. The composition of the chromatin-bound protein complexes and their temporally and spatially regulated recruitment influence gene expression. Recent findings are consistent with the concept that chromatin modifiers play an important role in regulating skeletal muscle gene expression and cellular differentiation.

Interplay between Metabolites and the Epigenome in Regulating Embryonic and Adult Stem Cell Potency and Maintenance.

The environment surrounding stem cells has the ability to elicit profound, heritable epigenetic changes orchestrated by multiple epigenetic mechanisms, which can be modulated by the level of specific metabolites. In this review, we highlight the significance of metabolism in regulating stem cell homeostasis, cell state, and differentiation capacity, using metabolic regulation of embryonic and adult muscle stem cells as examples, and cast light on the interaction between cellular metabolism and epigenetics. These new regulatory networks, based on the dynamic interplay between metabolism and epigenetics in stem cell biology, are important, not only for understanding tissue homeostasis, but to determine in vitro culture conditions which accurately support normal cell physiology.