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Dedifferentiation-Reprogrammed Mesenchymal Stem Cells with Improved therapeutic Potential

Original article from STEM CELLS

Recent studies have demonstrated that mesenchymal stem cells (MSCs) have the ability to differentiate into various kinds of cell types, including neuron-like cells in culture (Woodbury et al, Qian and Saltzman, Levy et al and Rismanchi et al) which has been further verified by transplantation experiments in various animal models of human disease. However, these studies have been hampered by reported low levels of cell persistence, neuronal differentiation in vivo and massive death of transplanted cells limiting their overall effectiveness and clinical use. Dedifferentiation is a process by which differentiated cells are reverted to an earlier, more primitive phenotype which confers an extended differentiation potential (Odelberg, Kollhoff and Keating) and previous studies by the authors of the study discussed herein demonstrated that by withdrawal of extrinsic stimulation, MSC-derived neurons are able to revert back to MSC morphologically (Woodbury, Reynold and Black and Li et al), but whether these dedifferentiated MSCs (DeMSCs) were similar to MSCs was unknown. This point is now addressed in the December issue of Stem Cells in a study (Liu et al) from the laboratories of Hsiao Chang Chan (Chinese University of Hong Kong, Shatin, Hong Kong) and Tingyu Li (Chongqing Medical University, Chongqing, China).

Initial rat MSC clones had normal characteristics and upon growth in a neural induction medium (MNM) following a pre-induction medium incubation, gradually expressed characteristics of neural cells over a 72 hour period and upon removal of the MNM neural differentiation medium, cells reverted back or dedifferentiated back to an MSC state (DeMSCs). However, they could henceforth be simply reverted back to the neural state by the addition of ATRA and bFGF to the culture medium, with nearly 100% of resulting cells being positive for the neural cytoskeleton markers NF-M and MAP2. Further, neuronal marker expression in DeMSCs was higher than the initial MSC clones suggesting that they retain some type of neuronal memory which aids neuronal redifferentiation. Analysis of excitability, the fundamental property of neurons, found a similar level of excitability for MSCs and DeMSCs and this rose during neuronal differentiation of both cultures but was significantly higher in the DeMSCs.   Further comparisons of MSCs and DeMSCs demonstrated that these two cell types had similar immunophenotypes and mesodermal differentiation potential with both cell types giving rise to mature osteoblasts, adipocytes, and chondrocytes after 21 day in vitro differentiation. Gene expression analysis showed few differences (1.5% upregulated and 3.0% downregulated by 2-fold or more in DeMSCs as compared to MSCs) and perhaps unsurprisingly, those genes that were upregulated in DeMSCs included imperative genes and growth factors for neural development or neurogenesis, while genes characteristic of MSCs remained unchanged. Additionally, analysis of Nestin and Musashi-1 as specific markers of neural stem cells and progenitors, demonstrated a higher level of expression in DeMSCs compared to MSCs suggesting that DeMSCs carried additional neuronal potentiality ready to be activated under appropriate conditions.

During these experiments, an increase in cell viability of DeMSCs was also observed. While spontaneous apoptosis levels were similar, DeMSCs has a survival advantage under conditions of oxidative stress (exposure to H2O2) which remained after in vitro culture and passaging. A focused apoptosis-related gene expression analysis found 13/84 genes differentially expressed between MSCs and DeMSCs, with Bcl-Xl, TNFa and Cidea being upregulated and Caspases, IAPs, Fas and ligand downregulated. The Bcl-2 family of proteins is the most prominent gene group involved in cell survival and while only the transcript of Bcl-Xl was enriched in DeMSCs compared to MSCs, protein expression of both Bcl-Xl and Bcl-2 was upregulated in DeMSCs compared to MSCs in response to H2O2. Strikingly, while both Bcl-Xl and Bcl-2 expression were inhibited in response to H2O2 in MSCs no change was observed for Bcl-Xl or Bcl-2 in DeMSCs, indicating enhanced expression of Bcl-2 family proteins might be responsible for their resistance to oxidative stress. The miRNA miR-34a has been reported to be involved in the regulation of apoptosis through direct targeting of Bcl-2 (Chang et al and Raver-Shapira et al) and indeed miR-34a expression was markedly increased in response to H2O2 in MSCs while there was no change of miR-34a detected in DeMSCs, allowing for increased Bcl-2 stability in DeMSCs. Interestingly, at the basal level, miR34a was more highly expressed in DeMSCs and as miR-34a has been associated with neuronal differentiation and this increased expression could be associated with the increased neuronal potential of DeMSCs. Correlative to this hypothesis, miR-34a expression was significantly increased upon transition of MSCs and DeMSCs to neuronal differentiation and ectopic overexpression of miR-34a in MSCs lead to a significant increase of neural stem cell marker genes which were also observed to be upregulated in the De-MSCs compared to MSCs.

The study then turned to the potential for DeMSCs to be used for therapeutic uses. The model system utilised primary cultures of hippocampus neurons (PHN) exposed to different concentrations of H2O2 for 2 hours followed by co-culture with MSCs or De-MSCs for 24 hours.   In general co-culture with the DeMSCs led to a larger number of viable PHNs indicating enhanced cell survival and DeMSCs were also shown to have a greater neuronal differentiation potential in this model than MSCs. Next, an in vivo rat model of neonatal hypoxic-ischemic brain damage (HIBD) was used to analyse the effect of GFP tagged MSCs or DeMSCs, transplantation following occlusion of unilateral common carotid artery leading to hypoxia. 7 days after transplantation, only DeMSCs could be detected, indicating improved cell survival, while immunostaining revealed that some GFP+ cells expressed differentiated neuronal markers NF-M or MAP2, indicating neuronal differentiation from the DeMSCs in vivo. MSCs have been implicated in promoting endogenous angiogenesis after injury, and so DeMSCs may be promoting increased angiogenesis in the ischemic region. Indeed analysis found that 7 days post-treatment, the number of CD31+ vessels increased significantly in the stem cell-transplanted groups with DeMSCs with even higher levels. Further, some transplanted DeMSCs were immunoreactive to CD31 suggesting that they may have been transformed into endothelial cells or formed fusion cells with pre-existing endothelial cells. Further analysis of the HIBD mice 2 months post-transplantation showed that all experimental mice had increased cognitive function, which was again more pronounced for the DeMSCs transplanted mice, whose results were comparable to non-injured control mice.

Stem cell transplantation has been shown to improve functional outcome in animal models of human disease but low in vivo survival and differentiation potential of the transplanted cells has limited their overall effectiveness. This study demonstrates that DeMSCs appear to represent a distinct population of stem cells with advanced neuronal potentiality, with survival and neuronal differentiation advantages over undifferentiated MSCs under both in vitro and in vivo conditions and were superior to MSCs in repairing brain injury with improved long term memory retention.



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STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.