To our knowledge, this is the first description of procedures necessary to take patient biopsies and generate NPCs, neurons, and glia through a pluripotent intermediate in the complete absence of either xenobiotics or feeder cells, with commercially available reagents. As new xeno-free media formulations, and even clinical grade feeders, are now regularly brought to market, it is possible that the conditions used here are not the only feasible methods to achieve this end. This is the first report to document the site of integration for the reprogramming factors after xeno-free reprogramming and demonstrate that such integrations can be benign. As a result, this work can serve as a starting point for clinical application of hiPSCs. The presentation here of clinical-grade SOPs could help to ensure that these methods are applied consistently and appropriately.
The procedures and SOPs outlined here are modular; therefore, as reliable methods are introduced to improve this protocol, including the use of nonintegrative methods to reprogram cells to the pluripotent state, new specific SOPs detailing these approaches can easily be integrated into the existing framework. Recent evidence demonstrated that human somatic cells can be reprogrammed to a pluripotent state by introduction of the reprogramming factors by protein, mRNA, miRNA, plasmid DNA transfection, adenovirus, and Sendai virus. Any of these nonintegrative methods could easily be incorporated into the existing protocols and SOPs described here.
Of course, future clinical application of hiPSCs may not require derivation under completely xeno-free conditions, as a clinical trial was recently initiated using derivatives of an hESC line that was initially derived with animal products and grown on murine feeders [2
]. However, the process by which hiPSCs would need to be “cleaned up” to make them GMP compliant takes significant resources and, more importantly, considerable time. Instead, with the framework provided here, the testing and quality control mandated by regulatory agencies could be streamlined.
In the future, it will be crucial to establish standard QC tests for pluripotent stem cells in clinical translation. However, the establishment of standard QC procedures is challenging because criteria need to be established for their identity, purity, safety, and genetic stability. We have used established standard immunostaining and FACS methods to verify their identity and purity. We have also established tests for adventitious agents to ensure that these cells are free from any contaminating microorganisms or their by-products. In supplemental online Figure 2, we outline our work flow to demonstrate how each step in the process relies on successful translation of the previous step, and where QC becomes critical in the decision to proceed.
Unfortunately, no universal standards have been established to date to compare human pluripotent stem cells or their derivatives. The currently available method to establish the pluripotent status of these stem cells is the teratoma assay. However, the immune-deficient status of the animals, the number of cells required to make teratoma, the number of repetitions, the site of injection, and the meaning for any negative results need to be predefined. It is possible that high-throughput surrogates for the teratoma assay and other QC criteria will be developed, perhaps even bioinformatic approaches using genomic data [22
]. Finally, characterization of the final transplantable cell product is at least as important as characterization of the undifferentiated stem cell itself for clinical translational purposes. Hence, significant effort and coordination will be required to establish appropriate QC tests, which should be customized for each type of specific final cell product.