Immortalization tools Shay/Wright-lab (version October 2013)

Background:

We believe that all not terminally differentiated diploid human cells have the potential to proliferate indefinitely, if their telomeres are maintained. In telomerase negative cells, telomere maintenance can be accomplished by ectopic expression of hTERT, as this is the limiting component of the telomerase holoenzyme. However, many cell types upregulate cell cycle inhibitors (such as p16) in response to inadequate cell culture conditions and therefore experience a telomere length-independent growth arrest (“culture shock”). We call the growth arrest due to inadequate growth conditions stasis (stress- or aberrant signaling- induced senescence), in contrast to replicative senescence, which is triggered by critically short telomeres. It is possible to circumvent stasis by adapting the culture conditions (depending on the cell type feeder layers, reducing oxygen, or media supplements may be sufficient), or by genetically blocking the cell cycle inhibitors (this has been done historically by viral proteins, such as SV40 large T, or more recently by knocking down p16, or overexpressing CDK4).

Immortalization strategies:

1)  Ectopic expression of hTERT: Some cells are readily immortalized by hTERT alone in conventional cell culture media (e.g. BJ foreskin fibroblasts in media X containing 10% serum). We have the following hTERT constructs:

  1. pBabe-hTERT
  2. pBabe-Lox-hTERT-Hygro (hTERT and hygromycin-resistance are flanked by Lox sites and can be excised by Cre-recombinase)
  3. pBabe-Lox-hTERT-Hygro-TK (hTERT, hygromycin-resistance and thymidine kinase are flanked by Lox sites and can be excised by Cre-recombinase; thymidine kinase can be used for negative selection for cells in which excision was not successful)
  4. lentiviral-hTERT-blast

2)  Growth in low oxygen: Most cells are not exposed to ambient oxygen levels (21%) in their native tissue, but to much lower levels (1-6%). Therefore, oxidative damage by ambient oxygen may trigger or contribute to stasis. We routinely cultivate many cell types in 2-5% oxygen (Wright & Shay 2006 Nat Protoc 1(4):2088-90).

3)  Addition of the following media supplements may help to prevent stasis:

  1. 20 mM HEPES
  2. 0.03 μg/ml Zinc sulfate
  3. 1.4 μg/ml Vitamin B12
  4. 0.055 μg/ml dexamethasone
  5. 0.1 mM N-(tert)-butyl hydroxylamine

4)  Overexpression of CDK4: Overexpressed CDK4 titrates away upregulated p16 (and possibly other cell cycle inhibitors that are upregulated and cause stasis, such as p27), without interfering with normal cell cycle control and differentiation processes. Thus, we have immortalized human myoblasts and bronchial epithelial cells using CDK4 + hTERT, maintaining the characteristics and differentiation potential of primary cultures. We have following CDK4 constructs:

  1. retroviral mouse-CDK4-neo
  2. retroviral mouse-CDK4-blast-Lox (from Minna lab, has not been tested in our lab, yet)
  3. lentiviral human-CDK4-hygro (not confirmed to work as efficiently as mouse-CDK4)