bp xvivo headerProduction of Retinal Progenitor Cells under Full-Time Optimized cGMP Conditions in the Barrier Isolator

recent paper in Nature’s Scientific Reports from the Budd Tucker’s lab at University of Iowa reported producing xeno-free clinical-grade retinal progenitor cells from iPSC in a barrier isolator with cGMP reagents and conditions. [1] This is very exciting news for the iPSC field. Continue reading to learn about this research involving production of retinal progenitor cells in optimized cGMP conditions.

The Findings
Wiley, et al reported:
• Overcoming patient age-related challenges in generating fibroblasts from skin punches using cGMP reagents and conditions
• The use of skin tissues from patients from 27 to 81 years of age to generate patient-specific iPSC
• The use of those iPSC cells to derive post-mitotic photoreceptor precursor cells which are optimal for retinal transplant
• Replacement of all animal-derived products with xeno-free reagents for reduced immunogenicity of the final cell product
• An efficiency of 50% of cell spheres forming cells suitable for transplant
• Generation of organoids from iPSC from patients with a NR2E3 gene mutation that results in skewing of mature photoreceptor fate. The development and final phenotype of the in vitro derived mutant organoids paralleled the fate of their in vivo counterparts.
• Most strikingly, the mature photoreceptor precursor cells were sufficiently stem-cell free that they did not generate tumors in mice
Personalized iPSC as a Strategy to Help the Blind See
The authors discuss how the eyes of patients with degradation of the retina lose the immune-privileged environment that normal eyes possess. Being able to generate patient-specific tissues obviates the need for a choice between blindness and a life of immunosuppression for affected patients.
Some groups advocate producing clinical iPSC from large banks of donors that span a range of HLA combinations. However, producing and characterizing all those cell cultures would be nearly as time-consuming and expensive as producing personalized cell cultures. This makes the efficient generation of cGMP transplantable photoreceptor precursor cells so important.
The protocol the Tucker group has developed seems to recapitulate the embryonic development of the human retina. While they are not the first group to report this in the growth of human retinal cells, it is interesting to note that oxygen and nutrient availability on the outside of cup-shaped multi-celled structures are thought to be key to the preponderance of more mature, post-mitotic photoreceptor cells in their final cultures.
cGMP Production of iPSC May Help Production of Other Tissue Types
“Regardless of the origin of the cells, the clinical-grade cGMP-compliant reagents and procedures described here have the potential to be widely applicable,” the authors state. iPSC can be used to generate any tissue type for almost any disease state. This is a big improvement in the published methods for producing human retinal precursor cells, but also a big step forward for the iPSC field in general.
Tissues for Research on Genetic Diseases
The finding that organoids made from patients with a NR2E3 gene mutation followed the same pathway and the same fate as the tissues in patients gives researchers a stable base from which they can try to cure the disease. It supports evidence that this gene may truly be responsible for what we see in patients and also opens up the possibility that if the mutated gene can be repaired, then personalized replacement retinal progenitor cells can be generated for these patients.
New Tissues from iPSC - Why Worry?
One of the tests of pluripotency, the ability of iPSC to generate different tissue types, is to inject the cells into mice and see if they form teratomas. These are tumors with lots of different mature tissues in them, even hair and teeth. It is proof that the iPSC can generate all of those different tissue types. If iPSC form tumors, that’s a good thing.
The downside? The iPSC form tumors.
Nobody wants to put tumor-causing cells into patients. There was a case reported of a patient in Italy who was part of a clinical trial in which nasal olfactory tissues were transplanted into the spine to try to regenerate nerve tissue. She later suffered the growth of a tumor there that produced mucus as well as bits of nerve and bone.
Researchers are also struggling with the possibility that if you insert new genes into cells to make iPSC, you may inadvertently cause damage to the cells’ own DNA that might cause them to form tumors after transplant. The human body can’t detect or reject tumors made from its own cells very easily.
The first-in-human clinical trials of iPSC-derived retinal cells in Japan were halted when mutations were detected in the second patient’s iPSC. This suggested possible tumor formation if they were used. Those trials still have not re-started pending revision of their cell production protocol.
No Tumors Formed with the Iowa Protocol
The Tucker group tried three different methods to generate tumors in mice. No tumors grew in any of the mice. This supports their conclusion that their cells were uniformly mature enough to not form tumors. Even more impressively, they were able to accomplish this without magnetic immune-panning or other techniques to try to pull immature stem cells out of the final cell product.
This is very encouraging for the iPSC community that has been struggling with tumorigenicity for 10 years. Once verified by independent studies, this will be a big step forward for the entire field, relieving the worry of tumor formation.
Full-Time Optimization of Cell Conditions
To get a new treatment to patients on a large scale, it has to be cost-efficient. Maximizing the yield and reliability of cell production could make a cellular therapy feasible and available to patients.
Taking the cytocentric point of view, protection from room air with a cGMP barrier isolator is essential for efficient therapeutic cell production. Full-time optimization of cell conditions, eliminating the variability that room air exposure introduces into cell production protocols, can improve cell yield.

Full-Time Protection of Patient Cultures
When precious patient-derived samples are at risk, full-time protection from contamination is also essential not only for patient protection, but for control of costs of cell production. After weeks or months in culture, to have to start over because of a contamination event would be not only costly, but would directly impact patients that quite literally have skin in the game.
The fact that contamination of patient cultures is completely preventable using available cytocentric technology and proper cGMP technique makes it even more incumbent upon the entire cellular therapy industry to use them.

1. Wiley, L.A., et al., cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness. Sci Rep, 2016. 6: p. 30742.

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alicia author iconAbout the Author

Alicia D Henn, PhD, MBA

Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.