Abstract:
The “Hayflick limit” is a “mitotic clock” and primary cells have a finite lifespan that correlates with telomere length. However, introduction of the telomerase catalytic protein component (TERT) is insufficient to immortalize most, but not all, human cell types under typical cell culture conditions. Originally, telomerase activity was only detected in cancer cells but is now recognized as being detectable in transit amplifying cells in tissues undergoing regeneration or in extreme conditions of wound repair. Here we report that in vitro low stress culture conditions allow normal human lung basal epithelial cells to grow for over 200 population doublings without engaging any telomere maintenance mechanism. This suggests that most reported instances of telomere-based replicative senescence are due to cell culture stress-induced premature senescence.
One Sentence Summary:
Human lung cells growing in reduced stress conditions can divide well beyond the Hayflick limit.
Main Text:
Introduction
Hayflick and Moorhead demonstrated that fetal lung fibroblasts grown in standard cell culture conditions in 21% atmospheric oxygen levels containing fetal bovine serum had a limited replicative lifespan in vitro of about 50-60 population doublings (PD), which became known as the Hayflick limit (1, 2). Since then others have demonstrated that oxygen sensitivity is one of the extrinsic factors limiting the replicative lifespan of both human (3) and murine fibroblasts (4).
Shortened telomeres correlated with and appeared to be a hallmark of human fibroblasts cultured in vitro for extended passages, but whether shortened telomeres had a causative role in replicative senescence was unknown (5). While a few cell types can be immortalized by just the ectopic introduction of TERT (catalytically active and rate limiting component of telomerase) (6), most human cell lines (under typical culture conditions) cannot be immortalized by exogenous TERT expression alone (7, 8). Thus, it remains to be determined if the Hayflick limit as originally described for fetal human lung fibroblasts is due to critically shortened telomeres or cell culture shock-induced premature senescence (9).
Telomerase is a conserved ribonucleoprotein enzyme complex (10) that uses an RNA template to reverse transcribe and add TTAGGG n DNA sequences at mammalian chromosome ends during DNA replication (11, 12). Initially, telomerase activity was only associated with advanced human tumors and cancer cell lines while most somatic tissues tested were telomerase negative (13). Further investigations detected telomerase activity in a subset of fast proliferating normal human cells, including hematopoietic tissues (14-17), skin (18, 19), hair follicles (20), and intestinal mucosa (21). Epithelial cell turnover in the lung occurs at a slower pace in the absence of damage. For example, ciliated tracheal cells have a half-life of six months and ciliated bronchial cells have a half-life of seventeen months in mouse lung (22).
While telomerase activity has not been previously reported in adult human lung tissue, it may be transiently expressed in basal progenitor cells during injury repair. The important role of telomerase in regeneration of adult tissue is underscored by the manifestation of genetic diseases in individuals with telomere spectrum disorders, ranging from bone marrow failure to idiopathic pulmonary fibrosis (23, 24). Lung basal progenitor cells have been shown to have long-term replicative capacity in vivo for lung repair and regeneration (25, 26), but senesceence in standard in vitro culture conditions after several passages.
Recently, we demonstrated that less stressful conditions for long-term expansion of primary human bronchial epithelial basal cells (HBECs) in vitro include co-culturing with an irradiated fibroblast feeder layer, ROCK inhibitor, and 2% oxygen (ROCKi conditions) (27). While differentiated lung epithelial cells are exposed to 21% atmospheric oxygen in vivo, basal lung stem cells residing near the basement membrane are exposed to much lower oxygen levels. Therefore, low oxygen culture conditions reduce the oxidative stress and DNA damage that occur in standard cell culture conditions that is not representative of in vivo conditions (28). For these reasons, the improved ROCKi conditions were modified from conditional reprogramming of cells (CRC) as originally described (29) to include 2% oxygen in addition to a change from standard epithelial cell proliferation F-media to a more defined Bronchial Epithelial Growth Media (BEGM) (27).
In CRC conditions and 21% oxygen, primary HBECs only grow for about 50 population doublings (29). Replacement of the fibroblast feeder layer with pharmacological inhibition of PAK1-ROCK-Myosin II and TGF-β signaling also extends primary HBEC proliferation in vitro to 50 population doublings, but accumulation of large cells preceded senescence and correlated with shortened telomeres (30).
We hypothesized that HBECs in ROCKi conditions would exhibit an extended lifespan compared to HBECs in standard culture conditions, but would senesce when telomeres reached a critically short telomere length or engage a telomere maintenance mechanism. Here we report the in vitro culture of primary HBECs well beyond the Hayflick limit without engaging a telomere length maintenance mechanism for over 200 population doublings (Figure 1A, blue line).
Rest at the source: https://www.biorxiv....0.1101/474270v2 (click on 'Preview .pdf).
