The advent of iPSC renewed hopes for cell-replacement therapy for PD. It is now possible to derive an unlimited number of pluripotent stem cell from either skin or other somatic tissues. Indeed, it has been shown that much like ES cells, iPS cells could be differentiated into DA neuron-like cells and these cells could provide symptomatic relieve in rodent models of PD (iPS cells 12
and ES cells 30
). In addition, because iPS cells are derived from the patients' own cells, they should not elicit an immunogenic response when transplanted back into the patients' brain, as is likely with transplantation of donor fetal tissues. Despite these significant advantages, there exists a significant hurdle before patient-derived iPS cells could be evaluated in human patients. Because of the use of genes such as Oct4
, which are known to promote carcinogenesis, there is a clear risk that there may exist a small number of cells that may become oncogenic during the iPSC induction process 12, 31
. Therefore, for any iPSC-derived cell-replacement treatment to be evaluated in humans, technological advances have to be made to reduce the carcinogenic risks of iPSCs significantly.
Direct-reprogramming approaches avoid much of the carcinogenic risks of the iPSC approach because differentiated cells (including both the starting and converted cells here) are less likely to form tumors when transplanted in vivo
. However, in its current form most direct-reprogramming approaches still use lentiviral vectors, which carry inherent risks of genome integration and inactivation of key tumor suppressor genes that may lead to tumor formation, albeit at much lower risks than the iPSC-associated methods. In the regard, promising alternative gene transduction approaches, such as RNA-based approach, have shown that lentiviral vectors could be replaced all together 32, 33
, thereby reducing much of the remaining risks for carcinogenesis in direct conversion of differentiated cells into DA neurons or other differentiated cell lineages.
Recently, there has been a wave of publications that report successful direct reprogramming of fibroblasts into neurons. The earliest of these is the successful conversion of mouse fibroblasts into induced neurons (iNs) using three transcription factors Ascl1
, and Myt1l
by Vierbuchen et al
. The same group also showed that the above three factors could convert human fibroblasts into neurons when combined with an additional transcription factor NeuroD1
. These cells showed morphological and electrophysiological behaviors that are typical of neurons. However, the exact identities of the converted neurons were not clear and they clearly lacked signatures of DA neurons. Using two of the above three transcription factors (MYT1L
) but with the addition of a microRNA (miR-124), Ambasudhan et al
showed that it is possible to convert human fibroblast cells into functional neurons. Again, these neurons, despite being functional, are not clearly defined in their subtypes. Similarly, Yoo et al
showed that it is possible to convert human fibroblasts into neurons with microRNAs miR-9/9*, miR-124 in combination with NeuroD2
. Qiang et al
showed that it is possible to directly convert fibroblasts from patients with Alzheimer's diseases into functional glutmatergenic forebrain neurons with transcription factors Ascl1
, and Myt1l
. Several of these protocols appear to confirm the importance of Ascl1
) in the reprogramming of fibroblasts into neurons, which was also proved important in our study.
More recently, two reports showed successful direct conversion of DA neuron-like cells in mouse 19
and human fibroblasts 19, 20
. Of these, Caiazzo et al
used transcription factors Ascl1
, and Lmx1a
. In the second report, Ascl1
, and Mytl
were used in combination with Lmx1a
. Both reports showed DA neuron-like cellular morphology and electrophysiology. However, our hiDA cells appear to be more morphologically mature when compared with those reported in those two reports. Furthermore, compared with our study, only limited in vivo
evaluation, in the form of injection and examination of brain slices shortly afterwards (4 days), was done in the report from Caiazzo et al
, and no in vivo
characterization was carried out in the report of Pfisterer et al
. No functional evaluation was carried out in rats with PD symptoms. In comparison, our study involves in vivo
functional studies in PD rats over an extended period of time.
In summary, results from this study suggest that our 5F cocktail can successfully reprogram human fibroblast cells into DA neuron-like cells. These cells behave like DA neuron cells both morphologically and functionally. Aside from adding to an increasing collection of successful and exciting direct cellular-reprogramming stories 17, 18, 19, 22, 34, 37, 38
, the main implication for our discovery is a potentially safe strategy to derive DA neuron-like cells for treatment of PD, a devastating neurological disease with no universally satisfactory treatment strategy currently available. Because these cells are of human origin and could be obtained with robust efficiency, we believe that they provide a promising source of DA neurons for evaluation as potential treatments for PD.