Cell culture has been a staple of biological and biomedical research since the inception of the first cell line, L-929, or “L cell”, derived from mouse subcutaneous connective tissue by WR Earle in 1948. Only four years later, GE Gey published the immortalized HeLa line, the well-known and widely used human cervical cancer cell line obtained from Henrietta Lacks. Cell lines are indispensable in multiple fields, including basic areas, such as genetics, epigenetics, developmental biology, to name a few, as well as more disease and application-oriented fields like cancer biology, neurodegenerative disease, and metabolic syndromes.
The cell culture toolbox contains numerous established methods to help answer research questions. Cell lines can be genetically manipulated to overexpress, knockdown, knockout, or knockin a gene of interest to determine its effect on phenotype, including inducible lines. They can also be modified to express tagged proteins, for example with a fluorescent protein for imaging and sensing applications or with short immunogenic epitope tags like FLAG and human influenza hemagglutinin (HA), e.g., for immunoprecipitation experiments and protein purification from cells. Cell lines are also amenable to reporter assays, a visible readout proportional to the various biological processes, for instance DNA repair, microRNA binding to mRNA targets etc. Cell lines were originally cultured in two dimensional (2D) monolayers, but are increasingly cultured as 3D spheroid and multi-layered organoids as more faithful tissue and organ mimics, with applications in transcriptome analysis and drug discovery. These basic and more advanced uses for cell lines have served as the foundation for countless discoveries and advancements in biology and biomedicine.
Recently, cell lines have been put to use for clinical applications and bioengineering of biopharmaceutical products, in addition to their more traditional roles in research. Although they have not yet made it into clinical practice, cell lines are being developed that are in clinical trials for cancer (e.g., with an immunogenic vaccine cell line or natural killer cell line NK-92), stroke, and neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Cell lines are also being developed from patient-derived skin tissue, by reprogramming the skin cells, i.e., fibroblasts, into induced pluripotent stem cells (iPSCs), which can then be differentiated into other cell types. This technology enables biomedical research on a genetic background derived from the patient, for disease- and person-specific data.
iPSC research applications in neurodegenerative diseases
Alzheimer's disease (AD), the most common neurodegenerative disease, affects 1 in 10 adults over the age of 65, and is dominated by cerebral cortex neuronal loss and dementia. Parkinson's disease (PD) is the second most frequent, damages dopaminergic neurons in the brain and leads to consequent movement defects. ALS is most characteristically defined by degeneration of upper and lower motor neurons, and results in muscle atrophy and loss of muscle control. “One similarity among many neurodegenerative diseases, including AD and ALS, which we study in our lab, is the prevalence of sporadic cases with no known genetic cause,” explained Dr Eva L. Feldman, the Russell N. DeJong Professor of Neurology and Director of the NeuroNetwork for Emerging Therapies at the University of Michigan. Dr Feldman has led a lifelong career investigating and understanding neurodegenerative diseases and translating basic discoveries into clinical applications. “Part of the challenge we’ve faced studying these diseases is the lack of a universal preclinical model,” Dr Feldman continued. “Most current ALS mouse models are based on known mutations, such as SOD1 (superoxide dismutase 1) or C9orf72 (chromosome 9 open reading frame 72). But, these mutations are not present in the vast majority of ALS patients, so the mouse models are not ideal.” To overcome this barrier, Dr Feldman and her team are developing ALS patient-derived iPSC lines. “These cells have the same genetic background as patients with the disease. We differentiate them into neurons, or iNeurons, and use these model systems to gain biological and mechanistic insight on the same genetic background as ALS individuals, a project led by Dr Benjamin Murdock in my lab.”
Another advantage of iPSC cell lines is that they can be differentiated into diverse other cell types. “Neurodegenerative diseases are extremely complex. Although neurons primarily degenerate, other cell types, such as the supportive glia, are likely involved,” explained Dr Benjamin Reubinoff, of the Hadassah Medical Center, a leading expert in stem cell line development. “We are differentiating iPSCs into other cells of the CNS (central nervous system), to investigate how they affect disease development.” In a recent publication in collaboration with Dr Feldman, Dr Reubinoff demonstrated that C9orf72 mutant astrocytes differentiated from patient-derived iPSCs contribute to ALS pathology by increasing oxidative stress, which damages motor neurons. “These are important advances”, Dr Reubinoff described of his research, “They indicate that targeting cell types other than motor neurons in ALS, and possibly other neurodegenerative diseases, could lead to potential treatments. iPSCs are making such discoveries possible.”
iPSC cell line technology is being used to shed light on the pathophysiology of other neurodegenerative diseases, including AD, PD, and frontotemporal lobar dementia, and other diseases, e.g., cardiac. Additional emergent applications involve drug discovery and personalized medicine and cancer vaccines.
Medical applications of cell lines in ALS and AD
Neuronal loss is a universal feature of neurodegenerative diseases, which has rendered them prime candidates for cellular therapies. “There is no cure for ALS. It is a relentless disease, and patients die within two to four years of their diagnosis,” Dr Feldman described of her experience treating ALS patients at the Michigan Medicine ALS Center of Excellence, which she directs. “The motor neuron degeneration leads to loss of muscle innervation and atrophy. Unfortunately, this ultimately ends in respiratory failure,” Dr Feldman elaborated of ALS pathology. “We’ve started clinical trials of stem cell line implants as a potential ALS therapy. The original idea was to replenish neuronal populations to slow, and ideally reverse, motor neuron decline. Instead, we are finding that the implanted cells support and nurture the remaining motor neurons, which slows disease progression.”
Dr Feldman and her colleague Dr Stephen Goutman utilize a stem cell line called NSI-566RSC. “As an established cell line, we can propagate and culture NSI-566RSC cells, so it circumvents the ethical concerns of primary embryonic stem cells. Of course one down side of using an established cell line is that it is non-autologous, meaning it does not match the patient antigen profile. So, we need to administer immunosuppressants to the ALS patient receiving NSI-566RSC cell line injections.” So far, the NSI-566RSC cell line has passed phase II clinical trials and the results look promising. “Although overall patient survival did not increase, we found that NSI-566RSC spinal cord injections improved functional performance and a composite functional/survival score in ALS patients, which could have important ramifications for their quality of life. We’re excited about the results and hope to advance to phase III trials,” Dr Feldman discussed of the trial results. Next on the horizon? “We are tackling other neurodegenerative diseases of course,” Dr Feldman elaborated of future directions. “Alzheimer’s disease is a big one for me. We have not entered clinical trials yet, but we do have a successful on-going preclinical program, which involves implanting a neural stem cell line, developed by our collaborator Dr Thomas Hazel, which expresses IGF1 (insulin-like growth factor 1), into AD rodent models.”
Dr Reubinoff is also highly engaged in the potential clinical applications of stem cell lines. “Stem cell lines have immense potential for regenerative medicine or as cell-based treatments for human diseases,” Dr Reubinoff explained of his stem cell line program. “We have established a pipeline at Hadassah Medical Center for generating human pluripotent stem cell lines that conform to good manufacturing practice (GMP). These cell lines are generated without animal products, i.e., are ‘xeno-free’, and do not contain animal pathogens that could potentially harm a patient. So, these stem cell lines are highly suitable for clinical applications,” he elaborated. “We envision ‘Regulatory-Ready’, meticulously developed and GMP-compliant pluripotent stem cell lines, which can be differentiated into neural cells, and possibly other cell types.” Dr Reubinoff and his team are testing the safety and efficacy of their stem cell lines for treating diseases in preclinical models of multiple sclerosis and cerebral palsy.
Though still categorically essential to basic research, cell line development has also traversed into the biomedical realm. “The current and future clinical applications of cell lines are bountiful,” Dr Feldman concluded, a sentiment echoed by Dr Reubinoff, who stated, “It is becoming increasingly essential to consider GMP compliance, safety, and regulatory criteria as stem cell lines become poised for clinical applications.”