Scientists find virally reprogrammed human iPSCs demonstrate abnormal gene expression, senescence, and apoptosis.

Scientists claim that the ability of nerve cells derived from human induced pluripotent stem cells (hiPSCs) to function in vivo may vary according to the method used to generate the iPSCs from adult cells. A team based in the Republic of Korea and the U.S. has found that neurons and neural precursor cells (NPCs) derived from virally reprogrammed iPSCs demonstrated residual expression of exogenous reprogramming genes, early senescence, and apoptotic cell death. In contrast, NPCs and dopamine neurons (DAs) derived from hiPSCs generated using a protein reprogramming technique were highly expandable, exhibited gene expression and other properties similar to those of the brain’s own dopamine neurons, and restored motor deficits in the rat model of Parkinson disease.

The researchers, led by Sang-Hun Lee, Ph.D., at Hanyang University’s College of Medicine in Korea, and Kwang-Soo Kim, Ph.D., at Harvard Medical School in the U.S., report their findings in the Journal of Clinical Investigation in a paper titled “Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.”

The majority of hiPSC lines have been generated using lentiviral and retroviral methods to deliver the reprogramming genes, but these techniques are known to generate multiple chromosomal integrations and possible genetic dysfunction, Drs. Lee and Kim and colleagues report. What hasn’t apparently been carried out to date are studies that systematically compare the physiological and differentiation properties of hiPSCs generated using different reprogramming methods. The researchers have previously developed a nonviral method for generating hIPSCs that directly delivers four reprogramming proteins to cells, by fusing the proteins to a cell-penetrating peptide.  

In their newly reported research, the team has compared the genetic and physiological characteristics of NPCs and DAs derived from human embryonic stem cells with hiPSCs generated using different viral and nonviral techniques. They then tested the cells in an in vivo model of Parkinson disease.

Eight hiPSC lines were generated by either lentiviral transduction, retroviral transduction, or the direct delivery of arginine-tagged reprogramming proteins. All eight hiPSC lines exhibited morphological features typical of hESCs, and expressed undifferentiated hESC markers and tumor recognition antigens. The researchers then optimized differentiation protocols for each cell line to trigger the production of NPCs and DAs.

The NPCs derived from all four lentivirus-based hiPSCs, but not the retrovirus- or protein-based hiPSCs, retained some residual expression of the exogenous reprogramming gene Oct4. In fact, further genetic analysis showed that in contrast to the silencing of endogenous gene expression that occurs during neuronal differentiation, expression levels of exogenous reprogramming factor genes in the lentivirus-based hiPSCs were increased during neuronal differentiation. “At present, it is not known why exogenous reprogramming genes exhibit differential silencing in lentivirus- and retrovirus-based hiPSC lines,” the authors note. 

NPCs from protein-based hiPSCs were, like the control hESC-derived NPCs, stably expandable for at least eight passages without any change in proliferation index. Strikingly, however, the researchers found, the lentivirus-based hiPSC NPCs abruptly stopped proliferating within 2–4 passages, and the retrovirus-based hiPSC-NPCs stopped proliferating within 3–4 passages. Dramatic increases in apoptotic cell death were also observed in passaged NPCs from both lentivirus- and retrovirus-based hiPSCs, but not in protein-based hiPSC-NPCs. The fact that some NPC lines could proliferate over at least a limited number of passages suggested that their eventual senescence was not related to continued expression of the exogenous reprogramming genes. Instead, studies showed that upregulation or induction of p53 expression preceded cellular senescence of both the lentivirus- and retrovirus-based NPCs during passaging.

Interestingly, although NPCs derived from retrovirus- and lentivirus-based hiPSCs displayed limited expandability and early senescence, neuronal differentiation appeared to occur normally in all hiPSCs, and optimized conditions allowed the efficient generation of NPCs and DA neurons. “These observations suggest that retrovirus- and lentivirus-based hiPSCs may be useful for a variety of biological and neurorepair studies, even if they are an unsuitable source of cells for future personalized medicine,” the authors conclude.

Further evaluation of the protein-based hiPSCs showed the cells could efficiently generate functional midbrain-like DA neurons in vitro, which exhibited mature neuronal morphologies, expressed mature marker genes, and demonstrated expected electrophysiological properties.

Implanting the protein-based hiPSC-derived NPCs and DA neurons into a rat model of Parkinson disease led to “moderate but significant” levels of functional recovery, under optimum donor cell conditions. “Our results suggest that protein-based reprogramming may be a viable approach for generating a patient-specific source of cells for treatment of PD and other degenerative diseases,” the authors conclude.

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