Stem cells, their derivatives, and fetal hippocampal neurons have recently received much attention as direct transplantation tools for epilepsy therapy [57
]. While functional integration of graft-derived cells is needed for restorative approaches, stem cell derived brain-implants can also be engineered to release therapeutically active molecules with the aim to provide therapeutic benefit by paracrine mechanisms. Finally, a combination of functional integration and paracrine drug delivery may provide improved benefits.
The first generation of adenosine secreting stem cells was engineered by a bi-allelic targeted deletion of the endogenous Adk
-gene in mouse embryonic stem cells (mESCs) [60
]. Using established protocols to induce neural differentiation, neural progenitor (NP) cells were generated. In these cells ADK-deficiency induced the release of therapeutic doses of adenosine, whereas corresponding wild-type cells did not release significant amounts of adenosine [60
]. Two studies were performed (in mice and rats, respectively) to investigate the therapeutic effectiveness of ADK-deficient mESCs [35
]. In both studies, the therapeutic cells were grafted into the infrahippocampal fissure, where they survived for at least 4 weeks and formed a dense cell cluster, likely a source for the paracrine release of adenosine. In both studies, a subset of cells migrated into the ipsilateral CA1, where those cells assumed a neuronal morphology and expressed the neuronal marker NeuN [35
]. In the first study [61
] (performed in rats), ADK-deficient mESC-derived NPs were implanted prior to the onset of electrical hippocampal kindling. Thus, kindling stimulations were performed in the presence of the grafted cells. In contrast to sham-controls or recipients of wild-type cells, recipients of adenosine releasing cells displayed sustained protection from generalized stage 4 or 5 seizures over 22 days following cell implantation. Overall, this study demonstrated a robust suppression of kindling epileptogenesis by adenosine-releasing mESC-derived infrahippocampal implants [61
]. In the second study, either ADK-deficient mESC-derived NPs, corresponding wild-type cells, or a sham procedure was administered to mice 24-hours after an epileptogenesis-triggering intraamygdaloid KA-injection [35
]. In this model of epileptogenesis spontaneous recurrent electrographic seizures normally develop within 12 days following KA-injection. Three weeks after KA-injection all animals were subjected to extensive EEG-monitoring of seizure activity. While all control animals experienced recurrent electrographic seizures, none of the recipients of the adenosine-releasing cells developed any seizure [35
]. In addition to the complete lack of seizures, recipients of adenosine-releasing cells were characterized by a significant reduction in astrogliosis and by normal levels of ADK expression [35
]. These results suggest a potential antiepileptogenic effect of adenosine-releasing brain implants.
The use of human embryonic stem cells (hESCs) for research and therapy has spawned much controversy in recent years. Potential strategies and pitfalls concerning hESC-based treatment strategies have recently been reviewed [62
]. hESCs isolated from the inner cell mass of human 4.5 day-old pre-implantation embryos have unlimited capacity for self-renewal in culture. Under defined culture conditions they can be directed to differentiate into any adult cell type. Apart from ethical concerns, significant scientific challenges need to be met before hESCs can safely be used in human patients: specific differentiation of the cells needs to be controlled by culture conditions, genetic modification, or selection procedures. Tumor formation needs to be excluded by depletion of tumorigenic cells or enrichment of non-tumorigenic cells and by the use of early passages and karyotyping to exclude genetic aberrations. Inflammation and graft rejection needs to be prevented by immunosuppression, the induction of immunotolerance, or somatic cell nuclear transfer. Although several protocols have been developed to direct hESCs into neuronal differentiation pathways in vitro
these cells have not yet been used in experimental epilepsy paradigms. Due to the availability of ethically acceptable and safer alternatives that can be tailored to patient-compatible or autologous approaches, it remains to be seen whether hESCs will be developed further for antiepileptic therapy. Eventually, the use of adult germ line stem cells as a source for functional neurons might provide a more promising alternative.
Adult stem cells constitute a versatile source for regenerative medicine and have – if patient-derived – the potential for personalized therapies and the advantage of autologous grafting without the need for immunosuppression. Adult stem cells can easily be derived from bone marrow, skeletal muscle, skin, or lipoaspirate. They normally form progeny according to their tissue of origin (i.e. skin stem cells will form skin); however, if directed experimentally into specified differentiation pathways they can form a wide variety of differentiated progeny including neurons. This might be beneficial, if functional integration or network interactions are desired. However, if paracrine drug delivery is the major goal, then the differentiation state of the transplanted cells is not of crucial importance as long as the cells will survive long enough to provide therapeutic benefit.
In order to efficiently engineer human adult stem cells for therapeutic adenosine delivery, we developed a lentiviral expression vector that expresses an inhibitory micro-RNA directed against ADK. This vector was highly effective in downregulating ADK in human mesenchymal stem cells (hMSCs). This strategy yielded hMSCs with up to 80% ADK-knockdown and was sufficient to trigger adenosine release [63
]. Using a mouse model of CA3-selective epileptogenesis, we transplanted ADK-knockdown hMSC, or respective control cells that expressed a lentivirus containing a scrambled RNA-sequence, into the infrahippocampal fissure at two different time points. When transplanted one week prior to KA-injection, the implanted ADK-knockdown cells provided robust neuroprotection and ameliorated acute KA-induced seizures [63
]. When transplanted 24 hours after the epileptogenesis-triggering KA-injection the ADK-knockdown cells reduced subsequent epileptogenesis [64
]. Three weeks after KA-injection, recipients of ADK-knockdown cells had significantly reduced seizure activity, significantly reduced astrogliosis and significantly reduced ADK upregulation [64
]. These findings suggest that hMSCs can survive at least three weeks within the infrahippocampal fissure. The implants exert therapeutic effects by paracrine adenosine-release in acute seizure paradigms and in chronic epilepsy. A caveat of these studies was that these experiments involved a combination of immunosuppression and cross-species transplantation (human into mouse); nevertheless, these data suggest that hMSCs derived from a patient could be engineered for therapeutic adenosine delivery in a personalized treatment approach.