Developmental potential of defined neural progenitors derived from mouse embryonic stem cells

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.     

bFGF induces an earlier expression of nephrogenic proteins after ischemic acute renal failure

Recovery from acute renal failure (ARF) requires the replacement of injured cells with new cells that restore tubule epithelial integrity. We described recently the expression of a wide range of nephrogenic proteins in tubular cells after ARF induced by ischemia-reperfusion (I/R) (Villanueva S, Cespedes C, and Vio CP. Am J Physiol Regul Integr Comp Physiol 290: R861-R870, 2006). These markers, namely, Vimentin, neural cell adhesion molecules (Ncam), basic fibroblast growth factor (bFGF), paired homeobox-2 (Pax-2), bone morphogene protein-7 (BMP-7), Noggin, Lim-1, Engrailed, Smad, phospho-Smad, hypoxia-induced factor-1alpha (HIF-1alpha), VEGF, and Tie-2, are expressed in a time frame similar to that observed in normal kidney development. bFGF participates in early kidney development as a morphogen involved in mesenchyme/epithelial transition, and it is reexpressed in the recovery phase of ARF. To test the hypothesis that bFGF can accelerate the regeneration after renal damage, we used recombinant bFGF and studied the expression pattern of the above described morphogens in ARF. Male Sprague-Dawley rats were subjected to 30 min of renal ischemic injury and were injected with bFGF 30 microg/kg followed by reperfusion. Rats were killed and the expression of nephrogenic proteins were analyzed by immunohistochemistry and Western blot analysis. In the animals subjected to I/R treated with bFGF, we observed a 12- to 24-h earlier and more abundant reexpression of the proteins Ncam, bFGF, Pax-2, BMP-7, Noggin, Lim-1, Engrailed, VEGF, and Tie-2 than the I/R untreated rats. In addition, we observed a reduction in renal damage markers ED-1 and alpha-smooth muscle actin. These results indicate that bFGF can participate in the regeneration process and suggest that the treatment with bFGF can induce an earlier regeneration process after ischemic acute renal failure.     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane

We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.     

The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity

Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.     

Differentiation of monkey embryonic stem cells into neural lineages

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.     

Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells

To examine the role of secreted signaling molecules and neurogenic genes in early development, we have developed a culture system for the controlled differentiation of mouse embryonic stem (ES) cells. In the current investigation, two of the earliest identified BMP antagonists/neural-inducing factors, noggin and chordin, were expressed in pluripotent mouse ES cells. Neurons were present as early as 24 h following transfection of ES cells with a pCS2/noggin expression plasmid, with differentiation peaking at 72 h. With neuronal differentiation, stem cell marker genes were down-regulated and neural determination genes expressed. Coculture experiments and exposure to noggin-conditioned medium produced similar neuronal differentiation of control ES cells, while addition of BMP-4 to noggin expressants strikingly inhibited neuronal differentiation. Transfection of ES cells with a pCS2/chordin expression vector or exposure to chordin-conditioned medium produced a more complex pattern of differentiation; ES cells formed neurons, mesenchymal cells as well as N-CAM-positive, nestin-positive neuroepithelial progenitors. These data suggest that, consistent with their different expression fields, noggin and chordin may play distinct roles in patterning the early mouse embryo.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors

Radial glia are among the first cells that develop in the embryonic central nervous system. They are progenitors of glia and neurons but their relationship with restricted precursors that are also derived from neuroepithelia is unclear. To clarify this issue, we analyzed expression of cell type specific markers (BLBP for radial glia, 5A5/E-NCAM for neuronal precursors and A2B5 for glial precursors) on cortical radial glia in vivo and their progeny in vitro. Clones of cortical cells initially expressing only BLBP gave rise to cells that were A2B5+ and eventually lost BLBP expression in vitro. BLBP is expressed in the rat neuroepithelium as early as E12.5 when there is little or no staining for A2B5 and 5A5. In E13.5-15.5 forebrain, A2B5 is spatially restricted co-localizing with a subset of the BLBP+ radial glia. Analysis of cells isolated acutely from embryonic cortices confirmed that BLBP expression could appear without, or together with, A2B5 or 5A5. The numbers of BLBP+/5A5+ cells decreased during neurogenesis while the numbers of BLBP+/A2B5+ cells remained high through the beginning of gliogenesis. The combined results demonstrate that spatially restricted subpopulations of radial glia along the dorsal-ventral axis acquire different markers for neuronal or glial precursors during CNS development.     

Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells represent an ideal source for cell engraftment in the damaged central nervous system (CNS). Understanding key signals that control ES cell differentiation may improve cell type-specific differentiation that is suitable for transplantation therapy. We tested the hypothesis that extracellular signal-regulated kinase (ERK) 1/2 phosphorylation is an early signaling event required for the neuronal differentiation of ES cells. Cultured mouse ES cells were treated with an all-trans-retinoic-acid (RA) protocol to generate neurally induced progenitor cells. Western blot analysis showed a dramatic increase in ERK 1/2 phosphorylation (p-ERK 1/2) 1-5 days after RA induction, which was attenuated in the presence of the p-ERK 1/2-specific inhibitor UO126. Phospho-ERK 1/2 inhibition significantly reduced the number of NeuN-positive cells and the expression of associated cytoskeletal proteins. In differentiating ES cells, there was increased nuclear translocation of STAT3 and decreased protein expression levels of GDNF, BDNF and NGF. STAT3 translocation was attenuated by UO126. Finally, caspase-3 activation was observed in the presence of UO126, suggesting that the ERK pathway also contributes to the survival of differentiating ES cells. These data indicate that ERK 1/2 phosphorylation is a key event required for early neuronal differentiation and survival of ES cells.     

Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures

Oct3/4 plays a critical role in maintaining embryonic stem cell pluripotency. Regulatable transgene-mediated sustained Oct3/4 expression in ES cells cultured in serum-free LIF-deficient medium caused accelerated differentiation to neuroectoderm-like cells that expressed Sox2, Otx1 and Emx2 and subsequently differentiated into neurons. Neurogenesis of ES cells is promoted by SDIA (stromal cell-derived inducing activity), which accumulates on the PA6 stromal cell surface. Oct3/4 expression in ES cells was maintained by SDIA whereas without it expression was promptly downregulated. Suppression of Oct3/4 abolished neuronal differentiation even after stimulation by SDIA. In contrast, sustained upregulated Oct3/4 expression enhanced SDIA-mediated neurogenesis of ES cells. Therefore, Oct3/4 appears to promote neuroectoderm formation and subsequent neuronal differentiation from ES cells.     

Gene expression profiling of mouse embryonic stem cell subpopulations

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.     

RaxL regulates chick ganglion cell development

RaxL is a paired-like homeobox gene involved in vertebrate eye morphogenesis. We examined RaxL protein expression patterns during chick retinal development in combination with ganglion cell markers including the RA4 antigen, cBrn-3, Islet-1 and neuronal type III beta-tubulin. Double-immunostaining demonstrated that downregulation of RaxL protein correlates with upregulation of ganglion cell markers in the ganglion cell layer (GCL). To explore this correlation in vivo, we performed gain- and loss-of-function experiments by electroporating retroviral vectors encoding wild-type and dominant-negative-RaxL into the optic vesicles of stage 10 chick embryos. Infection with virus expressing RaxL led to a 35% decrease in Islet-1-positive ganglion cells at E5.0 and a complete loss of ganglion cells at E15, with no effect on displaced amacrine cells in the GCL. When dominant-negative RaxL was expressed, the total number of cells in the GCL increased by approximately 40% at E5.0 but was reduced to 40% at E15, due to ectopic apoptosis in the GCL from E9 to E15. These results suggest that RaxL gives an inhibitory effect on ganglion cell development and that the loss of RaxL expression is required for maintenance of ganglion cells.