hTERT-immortalized Cell Lines: You Can Have Your Cake and Eat It Too

Brian Shapiro, Ph.D.

The ideal characteristics of an in vitro whole-cell model include: equivalence to in vivo physiology, genotypic and karyotypic stability, high proliferative capability, and the ability to be used at high passage. Does such a cell type exist? In this month’s blog, I will discuss human telomerase (hTERT)-immortalized cells (ICs), which possess all of these qualities¹.

hTERT ICs are derived from primary cells that are made to constitutively express the catalytic component of the enzyme hTERT. The expression of hTERT allows a cell to escape the replicative senescence that occurs in primary cells after a certain number of population doublings (i.e. the ‘Hayflick limit’)². This limit of proliferation is a consequence of the fact that each time a cell undergoes mitosis, its chromosomes shorten by 30 to 200 nucleotides. Telomeres, which are 8,000 nucleotide base pair noncoding repeat regions that cap either end of a chromosome, prevent the loss of coding DNA. Thus, an adult mammalian somatic cell may undergo between 30 to 50 cell divisions before it has lost its telomeres. hTERT is typically expressed in germ cells, fetal tissue, and some tumor cells, where it functions to protect mammalian reproductive capability, early development, and tumor growth, respectively³.

With the discovery of its telomere-extending properties, it was inevitable that someone would test hTERT’s ability to extend the lifetime of non-transformed cultured cells. Interestingly, the stable transfection of telomerase into somatic human cells has been observed to increase their lifespan 5-fold³. Moreover, primary cells that are induced to stably express hTERT retain many of their in vivo biologies at high passage, are diploid or near-diploid, and show gene expression similar to the parental cell¹. Furthermore, because hTERT ICs are derived from a single clone, there is limited lot-to-lot variability, which reduces inconsistencies among experiments.

hTERT ICs reveal their usefulness as controls in experiments designed to determine the pathogenesis of cancer. For example, hTERT-immortalized human bronchial epithelial cells (HBEC3-KT) have been implemented as an analog for normal cells in lung cancer studies because of their similarity to primary cells; they do not exhibit the hallmarks of cancer cells, such as forming colonies in soft agar or producing tumors when injected into mice. Also, because they are not transformed, HBEC3-KT are wild type for the tumor suppressor protein p53 and the oncogene KRAS^V12. Thus, HBEC3-KT have been used in experiments as controls against tumor cell lines with those common mutations. These studies have identified some of the pre-mRNA splicing regulators, such as heterogeneous nuclear ribonucleoprotein L and splicing regulator p30a, which are responsible for driving multidrug resistance in non-small cell lung carcinoma cells^4,5.

hTERT-ICs have been used to study the intracellular pathways that drive oncogenesis in other tissue types as well. For instance, human mammary epithelial cells (hTERT-HME1) have been utilized as controls in breast cancer studies. Like HBEC-3KT, hTERT-HME1 present with an untransformed phenotype, and have normal expression pattern of tumor suppressors and oncogenes. The studies utilizing hTERT-HME1 as controls have uncovered critical information in the pathogenesis of breast cancer, such as the sensitization capacity of BRMS1 gene in ATP-induced growth inhibition and apoptosis, as well as the role of various caveolin 1 mutations in breast cancer tumorigenesis^6,7.

While hTERT ICs provide researchers with a continuously proliferating source of physiological controls for their experiments, they also indicate their usefulness in generating 3D models of complex tissue. The ability to develop 3D culture models is a growing area for studying drug efficacy, toxicology of therapeutics, and organogenesis. These culture models enhance target validation studies and reduce the number of animals needed in toxicological studies. In one study performed by ATCC scientists, early and late passage co-cultures of hTERT-immortalized keratinocytes (Ker-CT) and hTERT-immortalized mesenchymal stem cells (MSCs) formed epidermis-like stratified structures that presented strata-appropriate markers of differentiation (such as keratin 1, keratin 14, and filaggrin). In addition, the Ker-CT and hTERT-MSC co-cultures, after undergoing a scratch test assay, displayed re-epidermalization of their wounds⁸.

hTERT IC 3D culture models are not confined to the epidermis; in another example of the ability of these cells to form physiological structures, hTERT-immortalized aortic endothelial cells co-cultured with hTERT MSCs formed tubular networks reminiscent of blood vessels. This variant of the co-culture model also demonstrated physiological responses to pro- and anti-angiogenic factors. ATCC scientists observed that vascular endothelial growth factor (VEGF) concentration-dependently stimulated vascular tubule formation. Interestingly, the formation of these tubules was inhibited by treatment with VEGF antibodies (unpublished) or suramin⁹. Thus, the fact that hTERT IC-based 3D culture systems show real tissue responses to physiological stimuli uncovers their potential utility in pharmaceutical testing and toxicology studies. (Note: If you are interested in seeing how these studies were performed you can view the epidermal and angiogenic poster presentations on the ATCC website. ATCC also has numerous hTERT-related materials, such as the hTERT Cell Culture Guide, recorded Webinar Presentations, and application notes, which can be found in the ATCC Cell Biology Learning Center.)

Overall, hTERT ICs possess the flexibility to be implemented as normal controls in genetic alterations studies, or create organotypic co-culture models. These cells exhibit the ideal qualities of a whole-cell model, including equivalence to normal physiology, genotypic and karyotypic stability, high proliferative capability, and the ability to use at high passage.

References

1.   Dickson MA, et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20(4):1436-47, 2000.

2.   Shay JW, Wright WE. Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol 1(1):72-6, 2000.

3.   Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279(5349):349-52, 1998.

4.   Goehe RW, et al. hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing. J Clin Invest 120(11):3923-39, 2010.

5.   Shultz JC, et al. Alternative splicing of caspase 9 is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation of SRp30a. Cancer Res 70(22):9185-96; 2010.

6.   Zhang Y, et al. BRMS1 Sensitizes Breast Cancer Cells to ATP-Induced Growth Suppression. Biores Open Access 2(2):77-83, 2013.

7.   Lee H, et al. Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (-/-) null mice show mammary epithelial cell hyperplasia. Am J Pathol 161(4):1357-69, 2002.

8.   Briley A, et al. Characterization of a three-dimensional (3D) organotypic skin model using keratinocytes and mesenchymal stem cells immortalized by hTERT. Poster presented at the annual American Society for Cell Biology meeting in Philadelphia, PA, 2014.

9.   Zou C, et al. Development and Characterization of an in vitro Co-culture Angiogenesis Assay System Using hTERT Immortalized Cells for High Throughput Drug Screening. Poster presented at the annual American Society for Cell Biology meeting in Philadelphia, PA, 2014.

Primary cells, in vitro models of physiological relevance

Brian A. Shapiro, Ph.D.

I have recently joined ATCC as a Technical Writer, and have 14 years of experience growing a wide variety of cells. In the coming months, I will address topics such as how to choose the right cell type for your experiments, cell line cross-contamination, and microbial contamination in this blog.

Biomedical scientists often rely on in vitro cell models for the study of human physiology and the pathogenesis of disease. Human primary cells (HPCs) are frequently disregarded as a choice for cell cultures, as they typically require more technical expertise to establish in the laboratory than other cell types and must be used in early passage. While HPCs may be challenging to generate, they have much in common with cells in vivo; therefore HPCs may be the perfect addition to your experiments.

HPCs can be used to represent normal tissue physiology as they retain many of the secretory, barrier, contractile, and other physiological functions of their in vivo condition. Further, HPCs usually have normal expression of tumor suppressor genes and proto-oncogenes; this allows HPCs to display normal cell cycle controls. The fact that HPCs possess gene expression patterns similar to cells in vivo indicates that they would be excellent controls in experiments using tumorigenic cell lines or cell lines derived from diseased tissue in the study of cancer, Parkinson’s disease, or microbial infection. 

Beyond their use as controls for pathological studies, HPCs can be applied in a wide range of experiments that examine normal tissue and organ physiology. For example, primary human bronchial/tracheal epithelial cells, when cultured in an air-liquid interface culture system, have been observed to form airway epithelium, which secretes mucus and exhibit waving cilia¹. In addition, more than one primary cell type may be co-cultured to form complex tissue systems. For instance, primary human neonatal foreskin keratinocytes cultured on fibroblasts differentiate into the four functional layers of the epidermis². Because organs are 3-D and boast multiple cell types, these co-culture and 3-D culture systems come close to mimicking physiological organ systems. The similarity to in vivo tissue and organ systems suggest that these 3-D culture systems have applications in toxicity, tissue development, carcinogenesis, cosmetic testing, and wound repair studies.

HPC maintenance is similar to that of any other cell line, thanks to the availability of optimized media and reagent formulations, affordable cell matrix solutions, and detailed protocols. An alternative to isolating the cells yourself is through ordering the cells from a well-known biological resource center, such as ATCC. ATCC supplies cells from a broad range of tissue sources and uses cell-specific markers to ensure a high level of purity post-isolation. In addition, ATCC tests HPCs for viability, as well as contamination by mycoplasma, bacteria, and yeast. Thus, when the HPCs arrive in your laboratory, you simply thaw and plate the cells in the appropriate culture medium. The HPCs can then be treated to stimulate the desired cellular responses at any time during the maintenance phase. 

Considering the required technical expertise, the expense, and the inaccessibility of source tissue, the addition of HPCs to a laboratory’s inventory of in vitro models may seem daunting. However, the rewards to your research are more than worth the trouble. Because HPCs are untransformed, have similar gene expression as the cells in situ, and exhibit similar physiologic function as in vivo cells, they are indispensable for a wide range of experiments that examine normal physiology or disease pathology.

References

1.  Berube K, et al. Human primary bronchial lung cell constructs: the new respiratory models. Toxicology 278(3):311-8, 2010.

2.  Gangatirkar P, et al. Establishment of 3D organotypic cultures using human neonatal epidermal cells. Nat Protoc 2(1):178-86, 2007.

The promise of MSCs

Carolyn Peluso, Ph.D.

 

In 2009, Charla Nash was the victim of a brutal animal mauling that left her without hands and with a face mutilated beyond recognition. Two years later, doctors attempted a face and hand transplant that took a surgical team 20 hours to perform. Sadly, the hand transplants failed almost immediately and had to be removed, but the face transplant succeeded, dramatically improving her quality of life. Her transplant was met with excitement from the medical establishment and the general community, which prompted invitations to appear on Oprah and the Today Show. Her transplant was considered something of a medical marvel and in fact, successful transplants of this kind (also known as a composite tissue allograph or CTA) are something of a rare bird. The first successful hand transplant occurred in 1998 and since then only about 200 patients have undergone the procedure worldwide¹ and with only limited success.  The rarity and poor success rate seems especially low when one considers that 28,000 patients undergo solid organ transplantations (e.g., heart, liver, kidney) with significant success in the just the US every year (United Network for Organ Sharing at www.UNOS.org).

In contrast to solid organ transplants, however, CTAs require the integration of multiple tissue types (i.e., muscle, fat, nerve, and highly-antigenic skin). The complexity of the transplant and the antigenicity of the skin tissue make rejection more likely than solid organ transplants¹. As a result, CTA patients are tied to the same onerous life-long burden of immunosuppressive therapy that solid organ recipients face, but in addition they face an increased risk of rejection complications.  Taking on the transplantation risks make sense for solid organ recipients who cannot survive without the transplant, but CTA recipients are generally healthy, aside from the missing limb². Thus, the procedure is about quality of life, not life-or-death, and the decision to proceed has to be balanced against the dangers of immunosuppressive therapy and rejection complications. Therefore, physicians need a way to reduce the immunological dangers of the CTA in order to offer it to patients on a wider scale; and this is where Mesenchymal Stem Cells (MSCs) may come into play.

MSCs are stromal cells that exist in tissues, such as the bone marrow and adipose and that can differentiate down multiple mesenchymal lineages (i.e., chrodrocytes, osteoblasts, adipocytes). This feature of MSCs has been successfully exploited to facilitate tissue reconstruction in orthopedic procedures³. In addition to their tissue regenerative capacity, however, MSCs exert important influence on both innate and adaptive immunity. For example, MSCs are able to affect innate immune function through various channels that include inhibiting the antigen-presenting function of dendritic cells, regulating HLA-G expression to block Natural Killer cell activity, and down-regulating IFN-g expression⁴. At the same time, MSCs affect adaptive immunity in part by mediating the nitric oxide system, and the expression of cytokines and growth factors⁴.  Clinically, the capacity of MSCs to affect the immune response in the transplant environment has been demonstrated in several studies. One of which, showed, using a randomized kidney transplant trial, that treatment with MSCs lowers the incidence of acute rejection, and opportunistic infection when compared to other immune-modulating therapies (i.e., anti-IL-2 antibodies)^{4, 5}.

Currently, researchers are applying the lessons learned by the solid organ transplant community to the specialized problems of the CTA procedure. For example, investigators need to establish a culture paradigm that optimizes the immune-modulatory potential of the cells, so they can overcome the high antigenicity of the skin graft component of the CTA – a problem not shared by the solid organ transplant community. To this end, researchers have shown in rat and primate models that treatment with MSCs can increase the survival time of skin grafts, although immunosuppression was still necessary and the rate of long-term tolerance was poor⁴. It’s a good first step, but there are still a lot of unanswered questions.  Investigators will need to develop an appropriate method of MSCs application, and they will need to understand how immunosuppressive drugs and MSCs interact so they can limit the negative influence of the former and improve the effectiveness of the later. The path is long, but the goal is in clear sight, and investigators are in active pursuit. It is exciting to think that someday, in the not so distant future, people like Charla Nash will be able to overcome life-altering tissue injuries in a common-place way, without being considered a medical marvel, thanks to MSCs.

1.            Schnider, JT et al. Site-specific immunosuppression in vascularized composite allotransplantation: prospects and potential. Clin Dev Immunol 2013, 495212 (2013).

2.            Antony, AK et al. Composite tissue allotransplantation and dysregulation in tissue repair and regeneration: a role for mesenchymal stem cells. Front Immunol 4, 188 (2013).

3.            Steinert, AF, Rackwitz, L, Gilbert, F, Noth, U & Tuan, RS. Concise review: the clinical application of mesenchymal stem cells for musculoskeletal regeneration: current status and perspectives. Stem Cells Transl Med 1, 237-47 (2012).

4.            Plock, JA, Schnider, JT, Solari, MG, Zheng, XX & Gorantla, VS. Perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation. Front Immunol 4, 175 (2013).

5.            Tan, J et al. Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. JAMA 307, 1169-77 (2012).

Once upon a time . . .

Carolyn Peluso, Ph.D.

. . . in a lab far, far away a postdoc sits nestled in among the test tubes and large, glass sequencing plates. Tapping his pen in time to the soulful sound of the Doobie Brothers, he analyzes 100 base pairs of hard-earned sequencing data, and dreams of an easier way. Years from now, as he tells his graduate students this story, they will unkindly cluck and roll with laughter. That hard-working post-doc of yesteryear was dreaming of next-generation sequencing, but he could never anticipate how it would revolutionize the way we approach cancer research and drug discovery.

Next generation sequencing refers to the high-throughput sequencing techniques that followed first generation Sanger sequencing. These technologies have led to the formation of large-scale sequencing initiatives that have generated a vast amount of actionable data. One such initiative is the Cancer Cell Line Encyclopedia (CCLE). The CCLE is a collaborative effort between Novartis and the Broad Institute that has released mutation data for 1,651 genes for nearly 1,000 cell lines. The CCLE research group used this data set to compare the copy number, expression pattern, and mutation frequency of tumor cell lines with primary tumors and showed that tumor cell lines are reasonably representative of their in vivo counterparts. Additionally, they used the sequencing data to predict that tumor cell lines harboring particular mutations are sensitive to specific classes of drugs¹.

Researchers are using this information, and the data from similar initiatives, to build better models to support basic research, and better platforms for screening potential drug candidates. ATCC is contributing to this effort by generating “sets” of tumor cell lines (the ATCC^® Tumor Cell Panels) that are annotated with mutational data, and arranged by tumor type, such as Pancreatic (TCP-1026™), Lung (TCP-1016™), and Breast (30-4500K™), or by commonly mutated genes like APC, EGFR, and BRAF.

Alone, these tools have the power to accelerate the research, development, and screening phase of drug discovery. The long-term hope, however, is to couple whole-genome sequencing to the transcript and epigenetic information from a single tumor sample. Having such information at their disposal, researchers will be able to develop better classifications for human cancers, and better, more personalized treatments. So, when they stop laughing, those graduate students should take a minute to thank their advisor. The long hours he spent in the lab, struggling for every base pair and thinking about a better way, were setting the stage for them to make huge strides towards a cure for cancer.

1.      Barretina, et al., (2012) Nature 483: 603-607

Choosing the best cell model for the job

Carolyn Peluso, Ph.D.

Our last several blog posts have described the causes of and the solutions to cell line contamination and misidentification. Hopefully, by now you are pretty confident that your cells are exactly what you thought. So on to the next step . . . how do you know that the cell line you have chosen for your experiments is a good model for the hypothesis you are testing?

Many investigators are asking the same question, and for good reason. One group looked at eight commonly used thyroid cancer cell lines, originally derived from thyroid tumors with diverse histological characteristics, representative of their individual states of differentiation. Microarray analysis of the cell lines revealed that all eight assume a similar dedifferentiated phenotype in vitro¹.  Thus, these cell lines may be useful if you are studying poorly differentiated forms of thyroid cancer. However, they may prove misleading if you are looking to answer questions about highly-differentiated thyroid tumors, and you are assuming that they have maintained the phenotypic character of the tumor from which they were derived.  

Another study compared the regulation of the retinoic acid receptor between an immortalized mouse Sertoli cell line (MSC-1) and primary Sertoli cells. They found that the cell line and the primary cells behave in a similar manner, indicating that for these studies at least the cell line is a good model for Sertoli cell function. When they expanded their studies to examine the immune privilege properties of Sertoli cells, they found that this is a property the MSC-1 cells do not share². Once again, this study demonstrates that cell lines are not always a perfect match for the disease or process under examination.

Clearly, some leg work is required when picking a cell line as a model system. First, it is always good to start with cells at early passage number. In general, cell lines are more likely to lose their parent cell character the longer they remain in culture. Second, before beginning a new set of experiments, the cell lines should be tested to ensure that, under normal conditions, the feature of the cell lines you are interested in studying matches up with the relevant primary cell. If you’ve tested that the cell line normally behaves like the primary cell, then alterations in behavior observed during your experiment are likely due to your manipulations and not a quirk of the culture conditions.  Cells grown in culture, and away from their in vivo environment, inevitably lose some of their in vivo character. As long as we appreciate this truth, and are diligent about performing control and proof-of-concept experiments, then cell lines will remain a valuable tool for modeling disease, and will continue to help scientists advance their research.

Next time we will take this discussion a step further, and look at how next generation sequencing is helping investigators generate better model systems for cancer research and drug discovery. So, until next time - we wish you good data and happy culturing,

ATCC Cell Biology