|Using iPSC Technology for Clinically Relevant Progenitors|
Two recent articles in Cell Stem Cell have highlighted a growing trend in induced pluripotent stem cell (iPSC) biology; direct reprogramming of somatic cells to tissue stem cells such as the direct reprogramming of fibroblasts into induced neural stem cells (iNSCs). Building on work from Sheng Ding and Marius Wernig, who first reprogrammed neural progenitors from fibroblasts (Kim et al and Lujan et al), the reports discussed herein from Frank Edenhofer and Hans R. Schöler now show the efficient derivation of iNSCs with extensive proliferative potential, a facet lacking from earlier reports, alongside neural morphology, expression profile, self-renewing capacity, epigenetic status and differentiation potential, as well as in vitro and in vivo functionality.
The first study from the laboratory of Frank Edenhofer began using the knowledge that NSCs are known to express Sox2, Klf4 and c-Myc (SKM), but not Oct4, so suggesting that fibroblast cells may be induced to NSCs through forced expression of SKM alone (Thier, Wörsdörfer and Lakes). The reprogramming process was undertaken using Oct4-GFP reporter mouse embryonic fibroblasts (MEFs) and a Dox-inducible Oct4 transgene, allowing for temporary Oct4 expression (the first 5 days only) during SKM expression. At 11 days post infection, rare neurosphere-like colonies emerged that exhibited neither iPSC-like morphology nor GFP fluorescence. To allow for more precise control over Oct4, direct delivery of Oct4 protein and mRNA was used, which also gave adherent GFP-negative neurosphere-like structures with b-III-tubulin+ processes emanating from the periphery. Colonies picked at day 18 were propagated in NS propagation medium leading to the outgrowth of NSC-like cells which could be grown in adherent culture.
Immunocytochemical characterisation of these cells (induced NSCs or iNSCs) found these cells to be positive for the NSC markers Nestin, Olig2, Sox2, and Blbp while RT-PCR confirmed transcription of Pax6, Blbp, and Sox2. Three independent experiments using 130,000 MEFs gave between 7 and 11 neurosphere structures each, of which 5 were isolated and 4 of which could be stably expanded to give iNSC lines which were grown for over 50 passages without changes in morphology, growth patterns or marker expression. Further, single-cell suspensions of iNSCs could be induced to form secondary neurospheres of which 60% could be expanded in adherent culture. All iNSC lines also exhibited complete silencing of all transgenes with induction of endogenous Sox2 but not Oct4, while all lines also carried genomic integrations of all SKM. Further comparisons were made using iNSCs, control NSCs, ESCs and MEFs through comparing global gene expression microarray data. As hoped, iNSCs were very similar to the control NSC line while although it was noted that there was subtle gene expression differences between the iNSC lines tested, they were all completely distinct from ESCs and parental MEFs. One of the iNSC lines was particularly similar to a foetal brain NSC line. Genes involved in NSC self-renewal and neural determination (Foxg1, Nes, Bmi1, and Olig2) were upregulated and fibroblast specific genes were downregulated (Col1a1, Col3a1, Dkk3, and Thy1) in iNSCs compared to fibroblasts, as expected, althougha subset of genes showed similar expression in MEFs and iNSCs suggesting some residual fibroblast epigenetic memory. Analysis or regional identity of iNSCs found that, similar to NSCs derived from the brain, iNSCs exhibit no specific regional identity but are more akin to a ventral fore/mid/hindbrain type.
Differentiation studies of iNSCs exhibited their ability to differentiate into astrocytes (GFAP+) through exposure to foetal calf serum, oligodendrocytes (O4+ and morphological attributes) through exposure to forskolin, triiodothyronine and ascorbic acid, and neurons (TIIIB+, NeuN+, and MAP2+) through exposure to BDNF in the absence of EGF/FGF. Phenotypic quantification of differentiated cells suggested that astrocytic and neuronal differentiation was favoured while oligodendroctyic differentiation was rare, further suggesting that iNSCs are similar to brain-derived NSCs (Conti et aland Glaser et al). Further characterisation demonstrated that all iNSCs lines gave rise to neurons with a GABAergic phenotype, with some able to express synaptic proteins. Analysis of membrane potential at three weeks differentiation also revealed the expression of a complex outward current pattern including inactivating and sustained components, reminiscent of neurons expressing both A-type and delayed rectifier potassium channels, while inward currents were only of small amplitude. However, 5 out of seven cells analysed were able to fire an action potential, while neurons also expressed glutamate and GABAA receptors on their surface, required for synapse formation. Finally, the in vivo development of iNSCs was studied by their suitability for glial cell replacement in neonatal myelin-deficient rats. Cells transplanted into the rat brain were detected 2 weeks later through staining of M2 and PLP, both of which are absent in this rat model, with positive cells exhibiting appropriate astrocytic morphology throughout a range of brain regions demonstrating that grafted iNSCs can survive and give rise to differentiated neural cells in vivo.
The next study from the laboratory of Hans R. Schölerused a slightly different method; using SKM but augmenting this initially by the expression of 8 neural specific transcription factors (Pax6, Olig2, Brn4/Pou3f4, E47/ Tcf3, Mash1/Ascl1, Sip1, Ngn2/Neurog2, Lim3/Lhx3; or POBEMSNL)(Han and Tapia et al). This however gave no NSC-like cells, leading the researchers to discard Ngn2 and Lim3 from their experiments which are associated with later stages of neural differentiation. Further analysis of transgene combinations allowed a minimal transgene set to be discovered – SKM with Brn4 and E47 (BSKME), which gave up to 5 NSC-like clusters at 4 to 5 weeks post-infections, which could be maintained as a cell line in culture and expressed suitable NSC markers. Such cells, again termed induced NSCs (iNSCs), could also be formed at a lower efficiency without E47 (BSKM), with both 5-factor and 4-factor iNSCs morphologically similar to control NSCs. Both iNSC types could be maintained in culture for over 130 passages and did not cause teratoma formation when injected into immunodeficient mice. However, while 4-factor iNSCs showed complete transgene silencing, transgenic expression of Sox2, Klf4 and Brn4 were detected in the 5-factor iNSCs although at levels lower than in control fibroblasts. Levels of endogenous factors were however similar to control NSCs, suggesting the initiation of the appropriate transcriptional pathways.
Transcriptome analysis of iNSCs, control NSCs and parental fibroblasts showed the expected conversion of the fibroblastic gene expression regime to an NSC transcriptional program, with the iNSCs and control NSCs being highly similar. However, early passage 4-factor iNSCs expressed intermediate levels of certain fibroblast specific genes, including Ctgf and Acta2, and showed low expression of some genes that are highly expressed in control NSCs. Late passage 4-factor iNSCs exhibited further down regulation of these genes to a level observed in control NSCs, while lower expressed NSC-associated genes were upregulated to levels similar to those observed in control NSCs. 5-factor iNSCs also showed expression of some fibroblast associated genes, but were more similar in their gene expression profiles to late-passage rather than early passage 4-factor iNSCs. This overall suggests a gradual change in transcriptional programs, perhaps through the dilution of epigenetic memory of the donor cells and the gradual reinforcement of the new cell fate. Analysis of DNA methylation of the second-intron of Nestin, which is unmethylated when expressed, demonstrated complete unmethylation in 4- and 5-factor iNSCs, as in controls NSCs, suggesting that epigenetic reprogramming had also taken place. Transcriptome analysis also allowed for the deduction of regional identity of the iNSCs, which suggested that iNSCs represent a more posterior neural cell type through the upregulation of posterior genes (Hoxa7, Hoxb7) and the downregulation of anterior neural markers (Foxg1, Emx1 and Otx2), and a more ventral regionalisation due to the expression of the ventral hindbrain gene Nkx6.1 and high expression of Olig2, a gene involved in the development of neurons in the ventral spinal cord.
Lastly, functionality and maturity of iNSCs was assessed. Both iNSC types could be differentiated into neurons that expressed sodium currents and that were able to generate single as well as multiple action potentials and astrocytes to a similar degree as control NSCs, but oligodendrocyte differentiation was impaired. Further analysis of these neurons found no major differences were observed in those generated from control NSCs versus iNSCs regarding their morphology, immunocytochemistry, and functional analyses. Transplantation of GFP-iNSCs into the subventricular zone of adult mice and analysis after two weeks demonstrated the presence of cells co-expressing GFP, the progenitor marker Nestin and the proliferation marker Ki67, although none co-expressing GFP and Sox2 suggesting that none of the cells remained as NSCs following transplantation and had differentiated in vivo or soon after engraftment. Indeed, Mash1, a marker for neural progenitors, was observed in some GFP+ cells, which were located at the edges of the grafts and some had migrated and integrated into the rostral migratory stream and expressed the neural markers Dcx and TuJ1. Glial (GFP+/GFAP+ and GFP+/NG2+ cells) and oligodendroctyic (GFP+/Olig2+ and GFP+/S100b+ cells) differentiation was also observed, suggesting that iNSCs can differentiate appropriately in vivo. Taking all these results together, we conclude that iNSCs have the potential to undergo differentiation both in vitro and in vivo into all neural cell lineages.
Overall these two reports show important progress in reprogramming, allowing the direct production of functional neural stem cells, an important cell type for potential clinical intervention. However, this work still requires to be replicated for human fibroblasts to show the potential relevance of this work to cell replacement in human disease.