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.