• Mesenchymal stem/stromal cells (MSCs) have a high potential for regenerative medicine and advanced therapies, with extensive research and over 1500 posted MSC clinical trials exploring their use in anti-inflammatory, oncology, and tissue regeneration applications.
• “Immortalization” of MSCs, particularly using telomerase reverse transcriptase (TERT), aims to extend the longevity of engineered cell populations, addressing primary cell senescence challenges to meet biopharmaceutical manufacturing demands.
• Advanced genome integration techniques, including CRISPR and/or non-viral vectors, might be key to developing MSC cell lines with prolonged doubling capacity for scalable bioproduction while mitigating the risks of cell transformation via traditional viral vectors.

If you want to incite a contentious discussion at your next dinner party, simply ask the question: If you were offered immortality, would you accept it? You may be surprised which of your friends believe mortality is a blessing or would dismiss it as a curse. You may also be surprised at the level of detail you need to give your friends about the situation. Would there be any penalty for immortality? Would you be the only immortal person in the world? You get the idea.

Unsurprisingly the question of immortality with regards to cells can also cause quite a heated debate. Mesenchymal stromal cells (MSCs) exhibit an extended but still-finite lifespan, having been shown to provide anti-inflammatory, pro-regenerative effects in vivo, and are investigated in over 1500 posted clinical trials.¹ Their versatility and amenability to large-scale culture in bioreactors is a major driver for their adoption by tissue engineers and developers of “secretome” ² or “MSC 2.0” ³ type clinical products. However, some may attest that MSCs, as a primary cell type, have been “cursed,” by their own mortality. This “curse” is acutely felt by those who wish to genetically engineer MSCs or use MSCs as a producer cell line of extracellular vesicles (EVs) or proteins. Cell engineering requires extended periods of selection and/or sorting, and hence, pushes the limits of a required cell  population doubling level, or PDL. ⁴

Imagine with me an MSC-like cell that has been genetically engineered to secrete extracellular vesicles containing an artificially displayed transmembrane ligand or scFv protein that targets a specific tissue of interest. ⁵ Or, if I may be so bold, an extracellular vesicle that is targeted and contains a therapeutic cargo. ^{6, 7, 8} How would one go about making such a supercharged MSC-EV producer cell?

To start, one would ideally generate what is known as a monoclonal cell line – an entire cell line born out of one single genetically engineered cell. This ensures that all cells have the same genetic modification of interest and will produce the desired engineered extracellular vesicles via a known genetic background and stable expression context. Creation of a monoclonal cell line requires a cell to undergo a multitude of divisions (i.e., =2^{Population Doubling Level}) ⁴ to reach a bankable population to be cryopreserved. Then, of course, the banked cells will have to undergo additional cell divisions after thaw for cell expansion and industrial-scale extracellular vesicle generation.

Table 1, above.  Assuming population doubling times of ~24h in high-performance medium, expansion of a single, engineered MSC clone into numbers sufficient for cell therapy doses or bioreactor runs can take many weeks and dozens of population doublings. However, as illustrated above, just enough doubling can yield massive numbers of cells, vastly exceeding global demand while satisfying early passage cryo-banking needs. ⁹ The difference in expansion capability between a primary cell and a transformed cell line underscores a need for “engineerable” primary cells that can reach high volumes without displaying hallmarks of oncogenic transformation.

Ideally, a cell will be able to undergo over 50 population doublings to get to this point (see Table 1, above). Herein lies a problem. Primary adult cells, such as bone marrow or adipose derived MSCs, can endure roughly 25-40 population doublings before senescence. ¹⁰ Cells from neonatal tissues such as umbilical cord can progress for somewhat longer, ¹¹ but will also ultimately arrest in their culture expansion. MSCs undergo telomere erosion with each subsequent division. When these telomeres reach a critical length (i.e., a “telomere crisis”), MSCs will initiate a signaling pathway leading to their senescence that serves to avert genomic instability. ^{12, 13, 14} Alternatively, there are many additional cell “stress” pathways that may be initiated and lead to senescence if an MSC senses trouble in its environment, such as oxidative, genotoxic, or metabolic stress, or inflammation. ^{15, 16, 17} To generate monoclonal cell lines, often a primary cell population can be engineered to suppress the native senescence pathways found in primary cells. ¹⁸

There are some ways to usurp native senescence machinery in the cell and force it to divide for longer durations, if not indefinitely. Remember those telomeres we mentioned earlier? Telomerase Reverse Transcriptase, or TERT for short, is part of a protein/RNA complex that lengthens telomeres. This combats the challenge of shortening telomeres that, with each cell division, marches cells towards fatal inevitable senescence. ¹⁹ MSCs have been successfully engineered to overexpress TERT and increase their lifespan in culture. ^{20, 21, 22} TERT is an appealing alternative to known viral immortalization genes associated with oncogenic transformation, such as SV40 large T antigen (SV40 LT), due to TERT’s presence in native cells and reduced pleiotropic effects. SV40 large T antigen prevents senescence via p53 and Rb pathway inhibition, disabling checkpoint-mediated senescence due to DNA damage, oxidative stress, or other insults outside of telomere erosion. ^{23, 24, 25}  In contrast to TERT, SV40 LT’s classic effects on cell phenotype are overt. ²⁶

Another consideration in the creation of immortalized cells is the method by which cells become immortalized. A common method is using lentivirus to transduce target cells with a gene of interest. Lentivirus is extremely effective at its job of gene insertion but comes with its own regulatory scrutiny when modified cells are re-infused. For one, even with titration, it is difficult to precisely control how many insertion events occur across a whole population of potential expanded clones. Regulators, such as the FDA, prefer to see less than 5 genome integration events per cell for ex vivo gene therapies. ²⁷ Additionally, it is imperative to ensure the lentivirus is replication incompetent in target cells. ²⁸ Even with these precautions, using virus for genetic engineering leaves cells vulnerable to insertional mutagenesis. ²⁹ These rare, but detectable events could ramify into actual consequences for human patients. ^{30, 31}  We are not able to “postmark” a donor gene to a specific landing site in the genome when we use viral integration methods, alone. The virus will, quasi-randomly, insert the DNA at various locations in the genome. This could lead to alternations in key genes, such as tumor suppressor genes, increasing the risk of an oncogenic transformation. Some alternatives to lentivirus include using a transposase, such as PiggyBac or Sleeping Beauty, or stable transfection using a linearized form of the donor gene. Homologous recombination directed integration, either alone ³² or in concert with a targeted genome editor activity,  ^{33, 34, 35} could pinpoint expression of the TERT or adjunctive gene products to a single locus in a “neighborhood” of chromatin conducive to long-term, stable expression—potentially a “genomic safe harbor.” ³⁶

No matter the path, immortalization of MSCs is certainly desirable for many emerging biomedical applications, and possible too. While RoosterBio will continue to promote the virtues of the mere mortal primary MSC, we certainly recognize that an “immortal” MSC would be one heck of a powerhouse for regenerative medicine applications—a “CHO-like” bioproduction cell for engineered extracellular vesicles/exosomes, ³⁷ mitochondria, ³⁸ and cloaked or retargeted virus capsids ³⁹ perhaps? Maybe there is room for mortals and immortals to coexist in this world…

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