Researchers find that these cells are primed to redifferentiate back into neuronal cells after transplantation.

Prompting expanded mesenchymal stem cells (MSCs) to undergo neuronal differentiation and then triggering them to dedifferentiate back to a more stem-like state results in a population of cells that exhibits enhanced survival when transplanted in vivo, and a greater ability to redifferentiate back into neuronal cells, investigators claim.

A team led by researchers at the Chinese University of Hong Kong was interested to see whether MSC-derived neuronal progenitor cells that were dedifferentiated (de-MSCs) would have different properties to the MSCs from which they were originally derived. Their findings showed that the de-MSCs displayed multipotent gene expression profiles similar to those of their unmanipulated MSC counterparts, but also upregulated the expression of genes required for neuronal differentiation and survival.

When MSCs and de-MSCs were then transplanted into a rat model of brain damage, the de-MSCs demonstrated a significant survival advantage, and led to comparatively greater levels of functional improvement in treated animals. The University’s Hsiao Chang Chan, Ph.D., and colleagues, suggest their findings could translate into more effective stem-cell based regenerative therapies. “It may provide a novel and clinical practical method to overcome low cell survival in cell-based therapy,” Dr. Chan states. “We are currently exploring other beneficial properties of the reprogrammed MSCs for other therapeutic applications.”

The team reports its findings in Stem Cell in a paper titled “Dedifferentiation-reprogrammed mesenchymal stem cells with improved therapeutic potential.”

Research has demonstrated that bone marrow stromal stem cells, also known as mesenchymal stem cells, have the capacity to differentiate into a wide range of cell types outside the mesodermal lineage, including neuron-like cells, the researchers report. Unfortunately, while transplantation studies using MSC-derived cells in animal models of diseases including Parkinson disease, stroke, cerebral ischemia, and spinal cord injury have shown beneficial effects, most have reported low levels of cell survival and neuronal differentiation in vivo.

Dedifferentiation, meanwhile, is a process that re-routes cell fate by reverting differentiated cells into an earlier, more primitive phenotype, with an altered genetic expression pattern that gives the cells a broader differentiation potential. While the process is common in, for example, amphibians that can regenerate complex body structures throughout life, research has only recently provided evidence that mammalian cells can undergo dedifferentiation.

Building on both their own and independent research, the Chinese University team looked more closely at dedifferentiated mammalian MSCs to investigate whether they may exhibit altered therapeutic potential compared with the MSCs from which they were originally derived.  

The researchers first isolated MSCs from rat bone marrow, expanded the cells and established monoclonal MSC clones. The resulting clones were then triggered to undergo neuronal differentiation. Once neuronal induction was established, the culture medium was changed again and cells were triggered to undergo dedifferentiation, reverting back to a stem cell state. The de-MSCs were passaged every 4–5 days, and induced to undergo neuronal redifferentiation by transferring washed cells to the appropriate modified neuronal medium.

Neuronal differentiation, dedifferentiation, and redifferentiation of monoclonal MSCs were confirmed by the concomitant upregulation and reversion of multiple neurogenesis marker genes as determined by PCR array, the researchers explain. Virtually 100% of the redifferentiated cells were NF-M and MAP2 positive. Interestingly, they add, while dedifferentiation from the neuronal to the stem cell phenotype was associated with a clear reduction in the expression of neuronal proteins, the expression of these neuronal markers in de-MSCs was higher than that in undifferentiated MSCs.

This suggested that de-MSCs retained some neuronal traits, and therefore presumably carried higher potential for redifferentiation into neurons. “Indeed, de-MSCs could undergo dedifferentiation with full expression of the neuronal markers,” the authors note. Moreover, the re-neurons derived from de-MSCs were significantly more hyperpolarized than neuronal progenitors derived from MSCs, indicating that they have a higher potential to develop into mature functional neurons.

Fluorescence-activated cell sorting (FACS) profiling indicated that de-MSCs retained an immunophenotype similar to that of undifferentiated MSCs, and both cell types could be induced in vitro to acquire typical characteristics of mature osteoblasts, adipocytes, and chondrocytes, indicating that the de-MSCs retain mesodermal potential. Gene expression profiling indicated that compared with MSCs, the de-MSCs demonstrated upregulation of about 1.5% of genes, and twofold or more downregulation of about 3% of genes.

Notably, the authors say that the most highly enriched transcripts in de-MSCs included critical and growth factor genes required for neuronal development or neurogenesis, although genes that were abundantly expressed in MSCs were also expressed at the same level in de-MSCs. And when the authors looked more closely at nestin and musashi-1, two genes that are widely considered as specific markers of neural stem cells and progenitors, they found that while only about 20–30% of undifferentiated MSCs expressed the genes, some 57–63% of de-MSCs were nestin- and musashi-positive cells.

The results thus far indicated that de-MSCs might represent a population of stem cells with increased neuronal potential, and in vitro studies also showed that compared with unmanipulated MSCs, they also demonstrated increased viability under conditions of oxidative stress (effected by treatment with H2O2). In fact, 13 out of 83 apoptosis-related genes were differentially expressed between MSCs and de-MSCs, with further analyses suggesting that the increased survival of de-MSCs under oxidative stress conditions might be due to enhanced expression of bcl-2 family proteins.

This finding led the team to look specifically at miR-34a expression in both basal and H2O2-treated MSCs and de-MSCs, because prior research has indicated that miR-34a is involved in regulating apoptosis through direct targeting of bcl-2. They found that miR-34a was markedly increased upon H2O2 treatment in MSCs whereas there was no change of miR-34a detected in de-MSCs, indicating that miR34a-targeted bcl-2 inhibition plays a role in the differential survival of MSCs and de-MSCs following H2O2 administration.

The miRNA investigation also threw up the finding that de-MSCs exhibited higher basal levels of miR-34a than MSCs. Given that previous studies had implicated this miRNA in neuronal differentiation, it was feasible to postulate that miR-34a could be associated with the increased neuronal potential of de-MSCs. In fact, they found that miR-34a expression increased over time as the neuronal differentiation of de-MSCs progressed. Of even more interest, they add, was the observation that miR-34a expression changed in parallel with both the neuronal differentiation and dedifferentiation process, providing a hint that miR-34a could be involved in regulating neurogenesis and contribute to the higher neuronal potential of de-MSCs. Supporting this notion was the finding that ectopic overexpression of miR-34a in MSCs resulted in a significant increase in three neural stem cell marker genes that were already upregulated in the de-MSCs.

To evaluate the therapeutic potential of de-MSCs, the researchers first exposed primary cultures of hippocampus neurons (PHN) to H2O2, and then co-cultured them with MSCs or de-MSCs. The H2O2 exposure caused the PHNs to undergo cytoskeletal disaggregation and axonal fragmentation. Co-culturing these damaged cells with MSCs or de-MSCs led to marked increases in the number of viable cells, but there were significantly more viable cells evident after co-culturing with de-MSCs than with MSCs.

Moving on to in vivo tests, the team focused on a rat model of neonatal hypoxic-ischemic brain damage (HIBD), induced in week-old animals by occlusion of unilateral common carotid artery. The HIBD rats were then treated using lateral ventricle injection of either GFP-tagged MSCs or de-MSCs. While both transplanted GFP-MSCs and de-MSCs were readily found near the injection sites at three days after transplantation, GFP expression could only be detected in de-MSC-treated animals by day seven after transplantation. A number of the transplanted cells had also migrated away from the injection site, and some of the GFP-de-MSCs expressed differentiated neuronal markers NF-M or MAP2.

The researchers then investigated whether the better survival of de-MSCs might be due to a greater ability to promote angiogenesis in the ischemic region. They stained ischemic brain tissue of both MSC- and de-MSC-transplanted rats for the endothelial marker CD31, seven days after treatment. The results confirmed that compared with untreated animals, CD31-positive vessels increased significantly in the stem cell-transplanted groups, but in comparison with MSC recipients, tissue from the de-MSC cohort showed much higher vessel density. “Of note, we observed some GFP-positive cells in de-MSC group were immunoreactive to CD31, indicating transplanted de-MSCs might have been transformed into endothelial cells or formed fusion cells with preexisting endothelial cells,” they state. “These results suggest that de-MSCs may be more pluripotent as compared to undifferentiated MSCs in promoting angiogenesis, which may contribute to their better survival in the ischemic region.”

This latter possibility was confirmed when the researchers compared the functional recovery of HIBD rats after treatment with MSCs and de-MSCs. The results of shuttle box tests showed that both treated groups significantly improved cognitive function, but as time went on, the functional improvements exhibited by the de-MSC-treated animals became obviously greater than those of the MSC-treated rats, and the tested behaviors were hardly different from those of control rats.

“The present study has characterized a previously undescribed dedifferentiation-reprogrammed stem cell population that exhibits enhanced cell survival and differentiation with improved therapeutic potential in vitro and in vivo,” the authors conclude. “With easy culture manipulation and low tendency of tumor formation, as compared to the more complicated genetic manipulation and higher risk of induced pluripotent stem cell forming teratomas upon transformation, de-MSCs may offer at least two advantages over iPS in cell-based therapy…Increased expression of bcl-2 family proteins and microRNA-34a appears to be the important mechanism giving rise to this previously undefined stem cell population that may provide a novel treatment strategy with improved therapeutic efficacy.”

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