The recent buzz in the iPS cell field  made me look at the problem of acquired immunogenicity by isogenic (autologous) cells in prolonged ex vivo culture. The immunogenicity of iPS cells didn’t come as a surprise to me, because I was aware of some data indicating possible acquired immunogenicity of autologous cells. Frankly, in the same time I came across a wonderful review by Chad Tang and Micha Drukker , which help me to understand and complete my view on this problem. I’m going to summarize the most important reasons, which could explain the phenomenon of acquired immunogenicity by autologous cells in vitro. All quotes have been taken from Tang-Drukker review.
So what are the mechanisms of acquired immunogenicity of autologous cells in prolonged cell culture?
1. Genetic modification
This is simple – gene transfer in cellular gene therapy could trigger immune response in the host. What we know about it so far is that immunogenicity could be caused by viral vectors  used as a therapeutic gene carrier. We don’t know whether or not non-viral vectors could potentially overcome this problem.
Well, immunogenicity caused by extensive gene manipulations seems to be expectable. But what if you just culture cells without gene transfer?
2. Xenoantigen incorporation in culture with animal-derived serum
This is a big time question guys! Because we are in a transition period from animal serum-based cell culture era to animal-free clinical-grade conditions for cell therapy. The problem is relatively well studied in pluripotent stem cell (PSC) culture:
One of the first pieces of evidence of this phenomenon was described by Martin et al . who discovered that human ESCs will uptake and aberrantly express the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) when cultured in standard conditions (e.g. ,on a mouse embryonic fibroblast (MEF) feeder layer in animal serum-containing media).
Before definitive conclusions can be drawn about the safety of Neu5Gc incorporation, further analysis including in vivo experiments should be conducted. We also stress that additional xenoantigens may be presented on PSCs as a result of in vitro exposure, and therefore, broader xenoantigen characterization efforts should be conducted prior to clinical applications.
Even though companies started to develop the series of animal-free cell culture products for cell therapy , it will take quite some time to optimize new conditions and standardize it for trials. Also I’d like to note that right now use of animal sera is not officially prohibited in cell manufacturing for clinical trials . The set of tests for viruses is applied, but issue of possible acquired immunogenicity is not investigated.
The bottom line: forget about any animal-derived products in your culture, if you develop cell therapies! We are done with it!
3. Aberrant antigen induction in serum-free prolong cell culture
What if we will get rid of all animal-derived products in our cell culture? Could it guarantee the absence of induced immunogenicity? The answer is NO, there is no guarantee. Apparently, cells can pick up some stuff from the culture medium, which can cause aberrant antigen expression:
However, animal products are not the sole factor that could produce aberrant antigen expression on PSCs. It is worth noting that even in the absence of animal-derived products, in vitro culture may still stimulate aberrant antigen expression as a result of nonphysiologically high concentrations of other media constituents. For example, recent studies have shown that prolonged exposure to high levels of serum factors normally found in humans can lead to aberrant antigen expression. In a series of papers, ectopic CD30 expression  has been shown to be induced from prolonged culture in animal-free knockout serum due to high ascorbate levels .
Again, this issue was studied in prolonged pluripotent stem cell culture. But I’d suspect that it could be applicable to any other type of cells.
4. Embryonic antigens expression on pluripotent cells triggers immune rejection
Because pluripotent stem cells are artificial entities of cell culture and don’t exist in adult human organism, their embryonic antigens could trigger immune response. Doesn’t matter embryonic stem cells or iPS cells. Markers of pluripotency are associate with early embryonic/ fetal development and carcinogenesis.
It is important to stress that the immune response against embryonic antigens would exist even in an isogenic setting.
In conclusion, these data indicate a degree of immunogenicity in fetal antigens resulting in a potential T cell mediated response against the differentiated products of PSCs. It is unlikely that the rapid pace of in vitro differentiation will precisely recapitulate the developmental processes that require months in utero. An immunological mismatch would therefore be created as embryonic antigens on therapeutic products encounter a mature immune system.
5. Acquisition of mutations and spontaneous transformation through extensive propagation in vitro
Both adult  and pluripotent  stem cells tend to acquire genetic and chromosomal abnormaliities during extensive propagation in culture. Spontaneous cell transformation could lead to immune response to cancer-associate antigens. However, the degree of this kind of immunogenicity remains to be investigated.
In addition to aberrant antigen induction from media contents, it is well established that genetic alterations accumulate due to prolonged in vitro culture.
Herszfeld et al. showed  a correlation between human ESC clones containing abnormal karyotypes with aberrant high CD30 expression.
Although a direct link between chromosomal lesions and immunogenicity has yet to be clearly demonstrated, given the frequency and number of lesions reported, it is likely that distinct PSC subclones may exhibit disparate immunological properties.
… isogenic PSC-derived transplants should not be considered non-immunogenic although they are genetically matched to an original donor.
Taken together, these data indicate that differentiated isogenic PSCs could potentially promote rejection processes unless immunosuppression measures are taken.
Nevertheless, we predict that the potential immunogenic reactions against isogenic PSC-derived transplants are subtler compared to those seen in allogenic directed at MHC and mHC mismatches. Therefore, we anticipate immunogenic reactions against isogenic transplants could be dampened via targeted therapy…
I’d like to emphasize that the same reasons for acquired immunogenicity cold be absolutely expected in adult stem/ progenitor cell culture.
What can we do about it?
It is clear that taking your own cells out of “body physiological context” could induce aberrant antigen expression and potentially trigger immune response. The possibility of aberrant antigen acquisition will correlate directly with time of cell culture and intensity of manipulations. So, first of all we have to admit and recognize this problem. Secondly, we should try to use fresh cells instead of cultured if they have nearly equal therapeutic benefit. But if we can’t avoid prolonged cell culture step, what else can we do?
- Exclude any animal-derived products and minimize time of cell culture.
- Minimize or avoid genetic manipulations ex vivo.
- Identify the markers, which could predict the possibility of rejection (CD30 could be good candidate ).
- Identify spontaneously transformed clones and exclude them from the graft.
- Identify residual pluripotent stem cells , contaminating graft, and eliminate them before transplant (For example via NK-cell lysis ).
- Use mild and selective immunosuppression in transplantation settings.
Finally, right now we still don’t know the clinical significance of this acquired immunogenicity. Maybe mild immunosuppression will be enough or will not required at all.