Victor F. Rafuse

A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.

Conversion of Mouse and Human Fibroblasts into Functional Spinal Motor Neurons

The mammalian nervous system comprises many distinct neuronal subtypes, each with its own phenotype and differential sensitivity to degenerative disease. Although specific neuronal types can be isolated from rodent embryos or engineered from stem cells for translational studies, transcription factor-mediated reprogramming might provide a more direct route to their generation. Here we report that the forced expression of select transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). iMNs displayed a morphology, gene expression signature, electrophysiology, synaptic functionality, in vivo engraftment capacity, and sensitivity to degenerative stimuli similar to those of embryo-derived motor neurons. We show that the converting fibroblasts do not transit through a proliferative neural progenitor state, and thus form bona fide motor neurons via a route distinct from embryonic development. Our findings demonstrate that fibroblasts can be converted directly into a specific differentiated and functional neural subtype, the spinal motor neuron.

Functional Properties of Motoneurons Derived from Mouse Embryonic Stem Cells

The capacity of embryonic stem (ES) cells to form functional motoneurons (MNs) and appropriate connections with muscle was investigated in vitro. ES cells were obtained from a transgenic mouse line in which the gene for enhanced green fluorescent protein (eGFP) is expressed under the control of the promotor of the MN specific homeobox gene Hb9. ES cells were exposed to retinoic acid (RA) and sonic hedgehog agonist (Hh-Ag1.3) to stimulate differentiation into MNs marked by expression of eGFP and the cholinergic transmitter synthetic enzyme choline acetyltransferase. Whole-cell patch-clamp recordings were made from eGFP-labeled cells to investigate the development of functional characteristics of MNs. In voltage-clamp mode, currents, including EPSCs, were recorded in response to exogenous applications of GABA, glycine, and glutamate. EGFP-labeled neurons also express voltage-activated ion channels including fast-inactivating Na+ channels, delayed rectifier and IA-type K+ channels, and Ca2+ channels. Current-clamp recordings demonstrated that eGFP-positive neurons generate repetitive trains of action potentials and that l-type Ca2+ channels mediate sustained depolarizations. When cocultured with a muscle cell line, clustering of acetylcholine receptors on muscle fibers adjacent to developing axons was seen. Intracellular recordings of muscle fibers adjacent to eGFP-positive axons revealed endplate potentials that increased in amplitude and frequency after glutamate application and were sensitive to TTX and curare. In summary, our findings demonstrate that MNs derived from ES cells develop appropriate transmitter receptors, intrinsic properties necessary for appropriate patterns of action potential firing and functional synapses with muscle fibers.

Polysialylated Neural Cell Adhesion Molecule Is Necessary for Selective Targeting of Regenerating Motor Neurons

It is well established that peripheral nerves regenerate after injury. Therefore, incomplete functional recovery usually results from misguided axons rather than a lack of regeneration per se. Despite this knowledge very little is known about the molecular mechanisms regulating axon guidance during regeneration. In the developing neuromuscular system the neural cell adhesion molecule (NCAM) and its polysialic acid (PSA) moiety are essential for proper motor axon guidance. In this study we used a well established model of nerve transection and repair to examine whether NCAM and/or PSA promotes selective regeneration of femoral motor nerves in wild-type and NCAM (-/-) mice. We found that regenerating axons innervating the muscle pathway and, to a lesser extent, cutaneous axons in the sensory pathway reexpress high levels of PSA during the time when the cut axons are crossing the lesion site. Second, we found that motor neurons in wild-type mice preferentially reinnervated muscle pathways, whereas motor neurons in NCAM (-/-) mice reinnervated muscle and cutaneous pathways with equal preference. Preferential regeneration was not observed in wild-type mice when PSA was removed enzymatically from the regenerating nerve, indicating that this form of selective motor axon targeting requires PSA. Finally, transgenic mice were used to show that the number of collateral sprouts, their field of arborization, and the withdrawal of misprojected axons were all attenuated significantly in mice lacking PSA. These results indicate that regenerating motor axons must express polysialylated NCAM, which reduces axon-axon adhesion and enables motor neurons to reinnervate their appropriate muscle targets selectively.

Motoneurons Derived from Embryonic Stem Cells Express Transcription Factors and Develop Phenotypes Characteristic of Medial Motor Column Neurons

Embryonic stem (ES) cells differentiate into functional motoneurons when treated with a sonic hedgehog (Shh) agonist and retinoic acid (RA). Whether ES cells can be directed to differentiate into specific subtypes of motoneurons is unknown. We treated embryoid bodies generated from HBG3 ES cells with a Shh agonist and RA for 5 d in culture to induce motoneuron differentiation. Enhanced green fluorescent protein (eGFP) expression was used to identify putative motoneurons, because eGFP is expressed under the control of the Hb9 promoter in HBG3 cells. We found that 96 ± 0.7% of the differentiated eGFP+ motoneurons expressed Lhx3, a homeobox gene expressed by postmitotic motoneurons in the medial motor column (MMCm), when the treated cells were plated on a neurite-promoting substrate for 5 d. When the treated embryoid bodies were transplanted into stage 17 chick neural tubes, the eGFP+ motoneurons migrated to the MMCm, expressed Lhx3, projected axons to the appropriate target for MMCm motoneurons (i.e., epaxial muscles), and contained synaptic vesicles within intramuscular axonal branches. In ovo and in vitro studies indicated that chemotropic factors emanating from the epaxial muscle and/or surrounding mesenchyme likely guide Lhx3+ motoneurons to their correct target. Finally, whole-cell patch-clamp recordings of transplanted ES cell-derived motoneurons demonstrated that they received synaptic input, elicited repetitive trains of action potentials, and developed passive membrane properties that were similar to host MMCm motoneurons. These results indicate that ES cells can be directed to form subtypes of neurons with specific phenotypic properties.

Transplanted Mouse Embryonic Stem-Cell-Derived Motoneurons Form Functional Motor Units and Reduce Muscle Atrophy

Prolonged muscle denervation resulting from motor neuron (MN) damage leads to atrophy and degeneration of neuromuscular junctions (NMJs), which can impart irreversible damage. In this study, we ask whether transplanted embryonic stem (ES) cells differentiated into MNs can form functional synapses with host muscle, and if so what effects do they have on the muscle. After transplantation into transected tibial nerves of adult mice, ES-cell-derived MNs formed functional synapses with denervated host muscle, which resulted in the ability to produce average tetanic forces of 44% of nonlesioned controls. ES-cell-derived motor units (MUs) had mean force values and ranges similar to control muscles. The number of type I fibers and fatigue resistance of the MUs were increased, and denervation-associated muscle atrophy was significantly reduced. These results demonstrate the capacity for ES-cell-derived MNs not only to incorporate into the adult host tissue, but also to exert changes in the target tissue. By providing the signals normally active during embryonic development and placing the cells in an environment with their target tissue, ES cells differentiate into MNs that give rise to functional MU output which resembles the MU output of endogenous MNs. This suggests that these signals combined with those present in the graft environment, lead to the activation of a program intended to produce a normal range of MU forces.

Neuroprotective properties of cultured neural progenitor cells are associated with the production of sonic hedgehog

Numerous studies have shown that abnormal motor behavior improves when neural progenitor cells (NPCs) are transplanted into animal models of neurodegeneration. The mechanisms responsible for this improvement are not fully understood. Indirect anatomical evidence suggests that attention of abnormal motor behavior is attributed, at least in part, to the secretion of trophic factors from the transplanted NPCs. However, there is little direct evidence supporting this hypothesis. Here we show that NPCs isolated from the subventricular zone (SVZ) of neonatal mice are highly teratogenic when transplanted into the neural tube of developing chick embryos and are neuroprotective for fetal dopaminergic neurons in culture because they release sonic hedgehog (Shh). In addition, the neuroprotective properties of NPCs can be exploited to promote better long-term survival of transplanted fetal neurons in an animal model of Parkinsons disease. Thus, cultured NPCs isolated from the SVZ can secrete at least one potent mitogen (Shh) that dramatically affects the fate of neighboring cells. This trait may account for some of the improvement in motor behavior often reported in animal models of neurodegeneration after transplantation of cultured NPCs that were isolated from the SVZ.

Easy and Rapid Differentiation of Embryonic Stem Cells into Functional Motoneurons Using Sonic Hedgehog‐Producing Cells

Directing embryonic stem (ES) cells to differentiate into functional motoneurons has proven to be a strong technique for studying neuronal development as well as being a potential source of tissue for cell replacement therapies involving spinal cord disorders. Unfortunately, one of the mitogenic factors (i.e., sonic hedgehog agonist) used for directed differentiation is not readily available, and thus this technique has not been widely accessible. Here, we present a novel and simple method to derive motoneurons from ES cells using readily attainable reagents. ES cells were derived from a mouse in which enhanced green fluorescent protein (eGFP) was linked to a motoneuron specific promoter. The cells were plated onto a monolayer of 293 EcR‐Shh cells that carry an integrated construct for the expression of sonic hedgehog (Shh) under ecdysone‐inducible control. To initiate motoneuron differentiation, 293 EcR‐Shh:ES cell cocultures were treated with ponasterone A (PA) and retinoic acid for 5 days. PA induces ecdysone, and thus drives Shh expression. To assess differentiation, putative ES cell‐derived motoneurons were studied immunocytochemically and cultured on chick myotubes for functional analysis. We found that ES cells differentiated into eGFP+ cells that expressed transcription factors typical of motoneurons. Furthermore, ES cell‐derived motoneurons were capable of forming functional connections with muscle fibers in vitro. Finally, when transplanted into the developing chick spinal cord, ES cell‐derived motoneurons migrated to the ventral horn and projected axons to appropriate muscle targets. In summary, this simple treatment paradigm produces functional motoneurons that can be used for both developmental and preclinical studies.

Neural cell adhesion molecule is required for stability of reinnervated neuromuscular junctions

Studies examining the etiology of motoneuron diseases usually focus on motoneuron death as the defining pathophysiology of the disease. However, impaired neuromuscular transmission and synapse withdrawal often precede cell death, raising the possibility that abnormalities in synaptic function contribute to disease onset. Although little is known about the mechanisms maintaining the synaptic integrity of neuromuscular junctions (NMJs), Drosophila studies suggest that Fasciclin II plays an important role. Inspired by these studies we used a reinnervation model of synaptogenesis to analyze neuromuscular function in mice lacking neural cell adhesion molecule (NCAM), the Fasciclin II vertebrate homolog. Our results showed that the recovery of contractile force was the same in wild‐type and NCAM−/− mice at 1 month after nerve injury, indicating that endplates were appropriately reformed. This normality was only transient because the contractile force and myofiber number decreased at 3 months after injury in NCAM−/− mice. Both declined further 3 months later. Myofibers degenerated, not because motoneurons died but because synapses were withdrawn. Although neurotransmission was initially normal at reinnervated NCAM−/− NMJs, it was significantly compromised 3 months later. Interestingly, the selective ablation of NCAM from motoneurons, or muscle fibers, did not mimic the deficits observed in reinnervated NCAM−/− mice. Taken together, these results indicate that NCAM is required to maintain normal synaptic function at reinnervated NMJs, although its loss pre‐synaptically or post‐synaptically is not sufficient to induce synaptic destabilization. Consideration is given to the role of NCAM in terminal Schwann cells for maintaining synaptic integrity and how NCAM dysfunction may contribute to motoneuron disorders.

Guidance of postural motoneurons requires MAPK/ERK signaling downstream of fibroblast growth factor receptor 1.

Identification of intracellular signaling pathways necessary for appropriate axon guidance is challenging because many CNS populations used to study these events contain multiple cell types. Here, we resolve this issue by using mouse embryonic stem (ES) cells that were directed to differentiate into a population of motoneurons that exclusively innervate epaxial muscles [medial median motor column (MMCm) motoneurons]. These ES cell-derived MMCm motoneurons, like their endogenous counterparts, express fibroblast growth factor receptor 1 (FGFR1) and selectively extend axons toward the epaxial trophin FGF8. Unlike wild-type MMCm motoneurons, FGFR1−/− MMCm motoneurons show guidance defects when transplanted into the neural tube of chick embryos. Furthermore, activation of FGFR1 selectively signals through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) for appropriate guidance in vitro, whereas overexpression of constitutively active MAPK/ERK in transplanted, or endogenous chick, MMCm cells causes guidance defects in vivo. These results indicate that MAPK/ERK activation downstream of FGFR1 is necessary for MMCm motor axon guidance and that ES cell-derived neurons provide an important tool for dissecting intracellular pathways required for axon guidance.