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Nestin Mediated NSC Purification



“Lineage-Specific Purification of Neural Stem-Progenitor Cells from Differentiated Mouse Induced Pluripotent Stem Cells”

From Stem Cells Translational Medicine.

While protocols for the differentiation of specific cell lineages from pluripotent cell types abound; the problem of the purification of these cells still remains unsolved.   Problems include the presence of remaining pluripotent stem cells in differentiated cultures which may cause tumourigenesis and the presence of other unwanted cell types.   To get around this problem, fluorescence-activated cell sorting (FACS) (Fukuda et al and Ladewig et al) or drug selection strategies (Li et al) have been proposed and used.   FACS purification of neural stem cells (NSCs) is difficult as many markers are not cell surface antigens (Lendahl et al and Kaneko et al), and so researchers from the laboratory of Tetsuo Sugimoto at the Kansai Medical University, Osaka, Japan have utilised a drug selection strategy, as is reported in Stem Cells Translational Medicine.   Using Nestin regulatory sequences in a rat model they report the successful isolation of pure NSCs using drug selection (Maruyama et al).

Initial analysis of various lengths of Nestin regulatory sequences in NE-4C cells (nestin-positive cell-derived neuroectodermal stem cell line) controlling luciferase expression found that the 257-base pair sequence within the nestin second intron (pN257H) was sufficient to function as an enhancer in NSCs.   Indeed transfection of pN257×2H, which includes two tandem copies of the 257-base pair sequence, showed further increases in luciferase expression.   Next, 2A peptide-based bi-cistronic expression vectors (Hasegawa et al) were constructed to allow for efficient expression of DsRed and the blasticidin S resistance gene (Bsd) from this regulatory sequence.   High expression of DsRed and Bsd were noted for vectors using pN257×2 to mediate expression compared to pNH which encompasses the entire Nestin second intron.   Further analysis of subcellular localization of DsRed and expression of Bsd, suggested that plasmids including Bsd-2A-DsRed construct (pNH-B2AD and pN257×2H-B2AD) were optimal for the generation of stable iPSCs using piggyBac transposon plasmids; alongside an enhancer-less plasmid (pH-B2AD) used to make a control clone.   G418-resistant iPSC colonies (which also expressed GFP under the control of the Nanog promoter) were generated and selected for by DsRed expression, which all showed genomic integration and the retention of parental iPSC-attributes.

In undifferentiated cultures, GFP-positive cells were also positive for DsRed in iPS(NH-B2AD) and iPS(N257×2H-B2AD) cells but only in 20% for iPS(H-B2AD).   This is to be expected as it is known that undifferentiated ESCs express nestin transcript (Cadiñanos and Bradley).   iPSC clones were then neurally differentiated using a modified serum-free defined medium-based selection protocol (Cai and Grabel, Okabe et al and Xu et al).   DsRed and Bsd expression in iPS(H-B2AD) cells was silenced during differentiation and was absent at day 10, while in iPS(NH-B2AD) cells, DsRed expression was observed at day 10, absent by day 13 and then was again observed at day 20 alongside Bsd expression.   However, DsRed expression in iPS(N257×2H-B2AD) cells was sustained till day 20, while Bsd was seen at day 13 and 20, suggesting that early DsRed expression is non-specific and later expression could be due to the Nestin enhancer.

Blasticidin S treatment of iPS(N257×2H-B2AD) cells for the 20 days of the differentiation protocol led to a decrease in cell number to 57% of non-treated number; although the Nestin-positive fraction rose from 47.5% to 66.7%, while Gata6- (endoderm) and Brachyury- (mesoderm) positive cell content decreased.   Flow cytometric analysis of viable cells found that differentiated iPS(N257×2H-B2AD) DsRed-positive cells increased from 56.4% to 63.5%.   Expression analysis of all cell types undergoing neuronal differentiation found a decrease in Oct4 expression, an increase in Sox2 (neural stem cell marker), nestin (ectoderm marker and neural stem cell marker) and musashi1 (neural stem cell marker) through to a high at day 20.   Blasticidin S treatment in iPS (N257×2H-B2AD) cells led to a 2.1 fold increase in in Nestin expression with little or no change in other markers.

The differentiation potential of blasticidin S-treated iPS (N257×2H-B2AD) cells was analysed after another 7 days of differentiation (day 27).   Nestin (NSC marker) was decreased slightly (to 43%) while 13% and 7.8% of MAP2- (mature neuron marker) and βIII- (early neuronal marker) positive cells with unambiguous neuronal morphology were detected at day 27.   The astrocytic markers S100β (3.8%) and GFAP (5.9%) were also observed at day 27 and while O4 staining was present, typical oligodendrocyte morphology was lacking.   Non-neural cells were found to make up less than 10% of total cell number.   mRNA analysis confirmed MAP2 and βIII-tubulin expression and demonstrated the increase in expression of tyrosine hydroxylase (monoaminergic neuron marker), glutamic acid decarboxylase 67 (GABAergic neuron marker), and Hb9 (motor neuron marker) at day 27 as compared with day 20, although choline acetyltransferase (cholinergic neuron marker) was unchanged.

In this report, the authors describe the usefulness of the Nestin enhancer sequences (N257×2) in the purification of NSCs from differentiating ESC cultures which have the potential to into neurons and glial cells; therapeutically important cell types.   The originality of this strategy was derived from the use of piggyBac transposon vector, however, the authors do note that their attempts to remove the plasmid transgene were unsuccessful; with only partial removal permitted meaning that further refinement of their constructs may be required if this strategy is to be therapeutically relevant.



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From Stem Cells Translational Medicine.

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.