What happens following pluripotent stem cell transplants into the brain? Are human IPS and ES cells going to function similarly in this context?
We recently published a new translational paper on the behavior of human pluripotent stem cells when transplanted into the 4dukt mouse brain in collaboration with my great UC Davis colleague, Dr. Veronica Martinez-Cerdeno.
Martinez-Cerdeno V, Barrilleaux B, McDonough A, Ariza J, Yuen B, Somanath P, Le C, Steward C, Horton K, Knoepfler P. Behavior of xeno-transplanted undifferentiated human induced pluripotent stem cells is impacted by microenvironment without evidence of tumors. Stem Cells Dev. 2017 Jul 10. doi: 10.1089/scd.2017.0059.
We are excited about this paper for a number of reasons. One of the things that makes it pretty unique is that we in parallel transplanted undifferentiated human iPSCs and human ESCs (hIPSC, hESC), whereas most studies pre-differentiate pluripotent stem cells. Even so we did not observe teratomas in the transplant recipients’ brains, which surprised us. It was also unexpected that we found that the mice did not strongly reject the transplants despite the animals being immunocompetent. The end result is that the mice survived without tumors and we got reasonable engraftment of both cell types and could compare their behavior.
You can see Figure 6 of our paper below, showing the fate of transplanted hIPSC where green staining highlights human cells and red staining is for the different indicated fate markers. Most human stem cells took on the appearance of NeuN+ neurons, while in contrast we did not observe human astrocytes. The numbers of human cells (and their fates) present in transplant recipient mice varied by region of the CNS although they were all injected stereotactically in the same location in the lateral ventricle, suggesting that microenvironment impacts transplanted cell behavior in key ways.
Why no tumors? In theory, immune rejection could have explained the lack of tumors. The mouse immune system perhaps killed the specific subpopulation of cells within the injected pluripotent stem cell cultures that had tumorigenic potential. It is also possible and I think more probable, since we observed a surprisingly high rate of fusion of the transplanted hIPSC or hESCs with host mouse brain cells, that this fusion eliminated tumorigenic potential.
We theorize that after human cell fusion with mouse cells, that nuclear fusion quickly followed since we did not observe multi-nucleated cells. The cell fusion we report is something that everyone studying cell transplants should look for in their studies. In theory transplanted stem cell fusion may not be entirely a bad thing as it could be exploited for drug delivery if we can better understand the mechanisms of fusion, but fusion can also potentially complicate apparent findings, especially if it isn’t on your radar screen.
Just because we didn’t observe tumors, doesn’t mean that undifferentiated human pluripotent stem cells would be safe to use clinically (I don’t think they would be) and other groups have reported teratoma in similar although not identical translational studies. In addition, we found in contrast to our human cell results that transplantation of mESCs did lead to teratoma or similar kinds of tumors such as one that appeared like a teratocarcinoma. It remains unclear why the mouse cells formed tumors while the human did not but xenotransplantation may lead to other effects such as the aforementioned cell fusion that impact biological outcomes.
There are a few limitations to our study on pluripotent stem cell transplants. For instance, it isn’t clear how a wider panel of hIPSC and hESC would behave, and some may be more predisposed to form teratoma. Also, we don’t know if the xeno nature of our transplants catalyzed the fusion and that leaves it unclear how much human stem cells would fuse with host differentiated cells following transplants. The clearest indicator of the rate of engraftment was by staining with a human-specific antibody and we also got some nice data by FISH, but while we tried a quantitative approach using qPCR to more precisely measure human gDNA present in the transplanted murine brain, this assay only worked in certain conditions. Most likely the qPCR assay sensitivity was limited by various factors including using fixed brain tissue DNA as starting material.
Overall, hIPSC and hESC behavior was quite similar in our study, further supporting the growing notion that reprogramming produces human cells generally equivalent to hESC, but a unique thing here in our study is that we found this similarity even within the transplant setting context in the brain (not just in a dish).
Many interesting follow ups could build on this work’s foundation including examining the impact of brain injury on transplanted undifferentiated hIPSC and/or hESC behavior. I’d be interested in any thoughts you have regarding the data in our paper on pluripotent stem cell transplants.
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