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Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors

From the June 2011 Issue of Stem Cells
Paper Commentary by Stuart P. Atkinson

The field of induced pluripotent stem cells (iPSCs) grows and expands with every passing day, fuelled by the seemingly graspable prize of a bona fide source of patient-specific pluripotent cells for regenerative medicine, be it for cell replacement therapy, drug screening or developmental studies. However underlying questions still remain pertaining to the cell type which undergoes reprogramming and ultimately their long term usefulness. In a new study (Kim et al) published in the June edition of Stem Cells, researchers from the laboratory of Rudolf Jaenisch at Massachusetts Institute of Technology, Cambridge, now try to answer some of these questions. Although iPSC have been generated from an increasing number of somatic cell types, the heterogeneity and the lack of markers for said cell types has not yet allowed the thorough study of the reprogramming of terminally differentiated somatic cells.

Kim et al have addressed this by studying reprogramming in terminally differentiated neurons isolated from mice bearing a CamKII-Cre allele and a LSL (LoxP-Stop-LoxP)-eGFP reporter allele. As the activity of the CamKII promoter is restricted to neurons with synaptic function, Cre becomes expressed and deletes the LSL cassette enabling the expression of eGFP to allow for the detection and selection of a pure post-mitotic neuron population from the adult forebrain and hippocampus. Tail tip fibroblasts from these mice were initially used to generate iPSCs using a polycistronic Dox-inducible lentiviral vector carrying the OSKM (Oct4-Sox2-Klf4-Myc) factors. The resulting iPSC were shown to be pluripotent and were injected into blastocysts to generate mice carrying both the neuronal tagging system and the integrated Dox-inducible reprogramming system to allow for secondary iPSC generation; simply by the addition of Dox to somatic cells isolated from these mice. In this case, purified terminally differentiated neurons from the adult forebrain and hippocampus.

Doubly modified mice at postnatal day 7 were used for neuronal culture, and eGFP expression was found only in the cortical plate, where post-mitotic neurons reside after migration from the subventricular zone during development. The vast majority of these cells also stained with NeuN, another marker for post-mitotic neurons, confirming the identity of these cells. Initial experiments showed that Dox treatment of eGFP-positive cortical neurons led to no proliferation, no repression of neuronal genes and no upregulation of any markers of pluripotency over a two weeks period. Further, treatment with Dox for 16 weeks led to no or little proliferation, cell death or induction of reprogramming. Dox-treated tail tip fibroblasts generated from the same mice however underwent robust growth, suggesting that OSKM alone is insufficient for the reprogramming of post-mitotic terminally differentiated neurons. This perhaps gives credence to the hypothesis that only a sub-population of cells are reprogrammed and, therefore, the observed low efficiency in many reprogramming experiments.

Rest and p53 inhibition where chosen as additional factors alongside OSKM to allow for iPSC generation. Rest is well known as a negative regulator of neuronal identity (Schoenherr and Anderson) while p53-inhibition allows the cells to overcome senescence-related signals generated by the reprogramming process (Menendez et al), thereby increasing the proliferative rate of the cells. Addition of Rest alone failed to allow for reprogramming to take place, however p53-inhibition resulted in iPSC generation. p53-inhibition in combination with Rest led to a 2-fold increase in alkaline phosphatase-positive colonies over p53-inhibition alone suggesting that the main “roadblock” to iPSC generation in these cells is the lack of proliferation while the repression of the neuronal identity is not sufficient to allow reprogramming alone but can enhance the process. Further analysis confirmed an increase in the proliferative rate and the repression of neuronal genes in cells undergoing reprogramming. Generated iPSC showed eGFP expression, confirming their neuronal origin, and also demonstrated Oct4, Nanog and SSEA1 expression, de-methylation of the Oct4 and Nanog promoters and the ability to form teratomas and chimeras. However, chimaeric mice showed early formation of tumours, perhaps due to genetic abnormalities accumulated by p53 inhibition, and therefore germline transmission was not assayed.

These data suggest that post-mitotic somatic cells are able to undergo reprogramming which leads to the generation of iPSC, but several points/questions still remain unanswered. 1) The efficiency of reprogramming is not mentioned in this study and direct infection of these cells with lentiviral constructs to generate iPSC was not undertaken, 2) Can other post-mitotic somatic cells be identified and purified by this means and reprogrammed? 3) Can this research be transferred from the mouse into a human cell population? and, 4) Can reprogramming occur without the need for p53-inhibition and the associated latent tumorigenicity of the cells which are generated? These questions aside, it is encouraging to see members of the iPSC field casting their eyes back in time and attempting to answer some of the basic questions surrounding iPSC which others seem to have dismissed or looked over. Perhaps more basic research such as this will in the end allow for the more efficient generation of cells with increased functionality, a problem which seems to be blighting current attempts to push iPSCs into the realm of therapeutics.



Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors.
Kim J, Lengner CJ, Kirak O, Hanna J, Cassady JP, Lodato MA, Wu S, Faddah DA, Steine EJ, Gao Q, Fu D, Dawlaty M, Jaenisch R.
Stem Cells. 2011 Apr 19

Silencing is golden: negative regulation in the control of neuronal gene transcription.
Schoenherr CJ, Anderson DJ.
Curr Opin Neurobiol. 1995 Oct;5(5):566-71. Review.

p53: guardian of reprogramming.
Menendez S, Camus S, Izpisua Belmonte JC.
Cell Cycle. 2010 Oct 1;9(19):3887-91. Epub 2010 Oct 9. Review.