Using a constitutively active form of the transcription factor MEF2C, we demonstrate production of the first stably transformed, ESC-derived, pure neuronal progenitor cell lines, designated MEF2CA-ESC-derived NPCs. We present evidence for the neuronal character of these cells after differentiation not only by neuronal markers but also from electrophysiological properties of these cells, both in vitro and in vivo. In vivo, we transplanted the MEF2CA-ESC-derived NPCs as a proof-of-principle therapy for stroke in a mouse tMCAO/reperfusion model and subsequently found improved neurobehavioral indices.
Stroke is a major cause of death and disability, but, despite intensive studies, few treatment options exist. Fetal brain tissue transplants have been shown to produce some recovery in animal models of stroke (Piccini et al., 1999
; Lindvall and Hagell, 2000
), but ethical considerations and a short supply of human fetal tissue limit this approach. Self-renewing and multipotent neural progenitor cells that give rise to neurons, astrocytes and oligodendrocytes have been found in the fetal and adult mammalian brain, including humans and rodents. Thus, the neural progenitor cell is likely to be a promising source of donor cells for brain transplantation. Embryonic stem cell cultures can be a nearly unlimited source of pluripotent cells, but the key to their successful use lies in the ability to control their differentiation and avoidance of tumor formation. Ideally, cells for transplantation into the brain would be differentiated to a stage where they are neurally restricted in their potential and expandable for production of large numbers. Also, it is necessary to provide mechanisms of control over the transplanted cells so that they can be directed toward the desired lineage, either neuronal or glial, and protected from induced apoptosis in the “hostile” environment of the mature brain. In the present report we describe results that show that expression of constitutively active MEF2 drives differentiation of mouse embryonic stem cells along a neuronal lineage, and that stable transformation of these ESC-derived neural progenitors with the MEF2C gene results in “neuronal
progenitor” cell lines (NPCs) that can be grown indefinitely in culture. We observed that the forced expression of constitutively active MEF2C in the neural progenitors has the effect of greatly biasing the differentiation pathway towards neurons and protecting the cells from apoptosis in vitro and in vivo after transplantation. It has recently been posited that new glial cells can originate from reactive gliosis, for example after a stroke, possibly obviating the need to specifically transplant this cell type (Buffo et al., 2008
) Further experiments will be required to confirm or disprove this supposition in our system.
Previously, using a combined molecular and bioinformatics approach, we found that a large number of neuronally restricted genes have MEF2 sites in their promoter regions and lack a TATA box (Krainc et al., 1998
; Okamoto et al., 2000
). This fact had first suggested to us that MEF2 might play a very important role in neurogenesis. Additionally, multiple MEF2 binding sites are located in the regulatory region of the Bcl-xL gene (S.-i. Okamoto and S.A. Lipton, unpublished observation). Bcl-xL is an anti-death member of the Bcl-2 family (Boise et al., 1993
; Frankowski et al., 1995
; Gonzalez-Garcia et al., 1995
; Krajewski et al., 1995b
; Krajewski et al., 1995a
; Roth et al., 1996
). Expression of such Bcl-2 family members in response to MEF2 activity may in fact protect new endogenous neurons following stroke, and permit further neurogenesis, suggesting a possible feedback loop (Zhang et al., 2006
). Combining these various lines of evidence, it was reasonable for us to hypothesize that MEF2 could be used as a transgene to protect neural progenitor cells during transplantation from apoptosis and to promote their neuronal differentiation.
We reasoned that for effective transplantation, forced expression of MEF2 should be restricted to the progenitor stage of differentiation to protect the cells and force commitment at this critical stage, but then allow the inherent differentiation programs to run once the cells were localized in the damaged brain. Nestin is an early filament gene that is expressed primarily in neural progenitor cells; the nestin enhancer sequence therefore was a driver that met our requirements. We therefore created constructs with the nestin enhancer sequence and thymidine kinase (tk) minimal promoter driving either EGFP alone for a control, or constitutively active MEF2C-IRES-EGFP. The present results demonstrate for the first time that constitutively active MEF2C (MEF2CA) regulated by the nestin enhancer not only promotes survival but also drives undifferentiated ES cells toward a neuronal phenotype in the absence of the influence of serum, growth factors or a feeder layer. Most of the ES cells in our hands expressed nestin following removal of LIF from the medium, concordant with the idea of a neural ‘default’ differentiation pathway (Tropepe et al., 2001
). Since the nestin/tk promoter drives our MEF2CA transgene, it was actively transcribed in these transiently transfected cells. However, in the absence of serum or growth factors, the improved survival and more pronounced bias towards neuronal development in cells expressing MEF2CA shows that this transgene was driving neurogenesis and protecting the cells from apoptosis. The anti-apoptotic activity of MEF2CA that we observed was as effective as that of transfected nestin/tk-Bcl-xL (), but under our conditions the Bcl-xL-transfected cells displayed a much stronger tendency to become progenitor or glial lineage cells (i.e., expressing GFAP in ), again emphasizing that in contrast MEF2CA expression promotes neurogenesis.
Our results with the transient transfection of ES cells suggested a strategy for generating stable cell lines predisposed to neuronal differentiation, which would be a potentially useful material for transplantation to correct neuronal damage due to degenerative disease or trauma. The nestin/TK-MEF2CA-EGFP transgene was stably integrated into ES cells and used to generate and select nestin-positive neuronal progenitor cell lines that recapitulated the transient transfection results. These EGFP marked cells represented proliferative neuroepithelial cells that expressed nestin and were maintained as “neurospheres” in culture medium containing FGF2 and EGF with minimal differentiation through at least eight passages in culture. Removal of the mitotic factors caused the cells to attach to substrate and differentiate. Downregulation of the nestin/tk-transgene (coupled to MEF2CA/EGFP or EGFP alone) occurred upon differentiation into neuronal or glial cell phenotypes. The nestin/tk-EGFP control cell lines generated mostly GFAP-positive glial cells with few neurons. In contrast, the proportion of cells expressing the neuronal markers TuJ1 and MAP-2 greatly increased in cell lines expressing the nestin/tk-MEF2CA-EGFP construct. Importantly, MEF2CA-expressing cells did not express muscle-specific myosin heavy chain (), as was the case with expression of MEF2CA in P19 teratocarcinoma cells (Okamoto et al., 2000
). Since MEF2 is also known to be an essential regulator of muscle development, this result shows that expression of the MEF2CA transgene in neural progenitors did not result in misdirected differentiation.
These MEF2CA-ESC-derived NPCs also produced more prominent neurite outgrowth upon differentiation. Differentiating the MEF2CA-ESC-derived NPCs on poly-l-lysine/laminin-coated glass cover slips produced cells that not only displayed neuronal proteins by immunocytochemistry but also electrophysiological characteristic of neurons, including TTX-sensitive fast sodium currents, and GABA- or glutamate-evoked currents.
In vitro studies can only provide a limited amount of information regarding the potential fates and effects of transplanted cells. It is well established that transplantation studies in mice require some form of brain damage to potentiate engraftment; mature cells transplanted into normal brains do not survive. In contrast, intracerebrally transplanted mouse ESCs, neural progenitor cells or v-myc immortalized neuroepithelial stem cells migrate toward a site of pathological injury (Villa et al., 2000
; Hoehn et al., 2002
; Modo et al., 2002
). Although the precise mechanism of migration of engrafted cells is unclear, it is possible that both the inflammatory response and intrinsic properties of the transplanted cells could play a role (Svendsen et al., 1996
; Svendsen et al., 1997
; Armstrong et al., 2000
; Li et al., 2000
; Chen et al., 2001
; Li, 2002
). To assess our MEF2CA-ESC-derived NPCs in vivo, we chose transplantation into the mouse tMCAOR stroke model, a procedure that produces reproducible ischemic damage to one cerebral hemisphere with distinct behavioral deficits.
The transplanted MEF2CA-ESC-derived NPCs that we characterized in the ischemic brains manifested a neuronal phenotype, as assessed by immunohistochemistry and electrophysiology. Moreover, the presence of synaptic currents suggested that the cells had started to integrate into the neuronal network of the host brain. The formation of functional synapses in this paradigm was important because the presence of MEF2 transcription factors have recently been shown to suppress synapse formation among cortical neurons ((Flavell et al., 2006
). In our paradigm, linking expression of MEF2CA to that of endogenous nestin, by use of the nestin/tk promoter to drive MEF2CA transcription, was a critical choice in our stable cell lines. Since nestin is only expressed at the NSC stage, once the MEF2CA-ESC-derived NPCs had differentiated into neurons, MEF2CA was downregulated coincident with nestin downregulation. Thus, after neuronal differentiation was initiated by MEF2CA, the transcription factor was apparently no longer necessary for continued neuronal development, and, in fact, its falling levels probably promote synapse formation in the cortex (Flavell et al., 2006
). Our results show that MEF2CA-ESC-derived NPCs are a viable source of transplantable cells for investigating cell therapy treatments for neurological brain damage. Importantly, due to our molecularly-directed differentiation of the cells to the stage of neuronal commitment, we never observed teratoma formation after transplantation of MEF2CA-ESC-derived NPCs (n > 50).
Additionally, behavioral studies after stroke on mice receiving MEF2CA-ESC-derived NPCs versus control neural progenitor cells showed that cell-replacement therapy had a positive effect. Extinction of the fear response requires learning in the prefrontal cortex to extinguish the conditioned behavior. We found that transplantation resulted in significant improvement of this behavioral paradigm. These results imply that transplantation of progenitor cells expressing MEF2CA made a significant contribution to the recovery of mice following ischemic brain damage, either through creation of new synaptic circuits, secretion of trophic molecules or a combination of the two.
Taken together, this report shows that forced expression of constitutively active MEF2C can keep neural progenitors alive while downstream differentiation events are directed along a neuronal lineage, and that such cells can be effective cell therapeutic agents in the treatment of ischemic brain injury. The results further suggest that MEF2C may possibly be permissive or even instructive for at least a component of the neurogenic program, and the so-called ‘default’ pathway to neuronal development may require MEF2 transcriptional activity since dominant negative MEF2C resulted in progenitor cell death. MEF2 is clearly not the only important transcription factor in neuronal differentiation, but it may represent a branch point in the pathway distinguishing ‘neural’ progenitor from ‘neuronal’ progenitor cell, at least in mouse cells. With this as a starting point, we have shown that mouse embryonic stem cells, treated as we have described, can be indefinitely expanded at a neuronal lineage-restricted stage of differentiation, where teratoma formation is no longer a threat. These MEF2CA-ESC-derived NPCs can be successfully used for cell-replacement therapy of neurological pathologies in an animal model system. Currently, a similar strategy is being tested in human ES cells as well.