J. Matthew Velkey

Glutamatergic Neuronal Differentiation of Mouse Embryonic Stem Cells after Transient Expression of Neurogenin 1 and Treatment with BDNF and GDNF: In Vitro and In Vivo Studies

Differentiation of the pluripotent neuroepithelium into neurons and glia is accomplished by the interaction of growth factors and cell-type restricted transcription factors. One approach to obtaining a particular neuronal phenotype is by recapitulating the expression of these factors in embryonic stem (ES) cells. Toward the eventual goal of auditory nerve replacement, the aim of the current investigation was to generate auditory nerve-like glutamatergic neurons from ES cells. Transient expression of Neurog1 promoted widespread neuronal differentiation in vitro; when supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), 75% of ES cell-derived neurons attained a glutamatergic phenotype after 5 d in vitro. Mouse ES cells were also placed into deafened guinea pig cochleae and Neurog1 expression was induced for 48 h followed by 26 d of BDNF/GDNF infusion. In vivo differentiation resulted in 50–75% of ES cells bearing markers of early neurons, and a majority of these cells had a glutamatergic phenotype. This is the first study to report a high percentage of ES cell differentiation into a glutamatergic phenotype and sets the stage for cell replacement of auditory nerve.

Development of the coronary blood supply: Changing concepts and current ideas

Earlier views of the development of the coronary vasculature included angiogenic budding and growth of arteries from the aortic sinuses and veins from the coronary sinus. The current concept begins with the establishment of the epicardium from the proepicardial organ, an outgrowth of the dorsal wall of the pericardial cavity. Capillaries form in a subepicardial mesenchymal population, extending as a plexus toward the truncus arteriosus and the atria. Multiple vessels grow from a peritruncal ring of capillaries, preferentially invading the newly formed aorta. In a process involving apoptotic changes of both the aortic wall and the invading capillaries, orifices open at the level of the aortic sinuses. Smooth muscle cells and pericytes, recruited from the surrounding mesenchyme, contribute to the vessel walls, and the definitive coronary artery pattern is established. Similar events are occurring on the venous side of the coronary circulation, following a slightly earlier time course. Multiple factors govern this process, including VEGF and FGF‐1 stimulating vasculogenesis and angiogenesis, and the angiopoietins and their tyrosine kinase receptors modulating interactions between endothelial cells and the mural components. As remodeling of the capillary plexus and the coronary orifices progresses, TGFβ released by apoptotic cells or from other sources likely modulates VEGF and FGF‐1, and also contributes to further apoptotic changes. A better appreciation of the controls of the mechanisms of coronary vessel development may direct further research in the prevention of arteriosclerosis and ischemic tissue injuries. Anat Rec (New Anat) 269:198–208, 2002.

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.

Apoptosis during coronary artery orifice development in the chick embryo

Previous studies regarding the development of proximal segments of the coronary arteries in the chick have demonstrated that these vessels do not develop as angiogenic outgrowths from the aorta. Rather, the proximal segments of the coronary arteries arise from a peritruncal capillary plexus in the epicardium that coalesces around the aortic and pulmonary outflow tracts. Vessels from the peritruncal plexus grow toward and attach to the aorta at about Hamburger and Hamilton (HH) Stage 32 to establish the definitive coronary circulation. Currently, little is known about the process by which patent connections are established between these peritruncal vessels and the aorta. The hypothesis that apoptosis is involved in the formation of the coronary artery orifices was tested in the present study. Aortic and periaortic tissue from HH 29–35 chick embryos was examined using routine light and electron microscopy and TUNEL assays. Apoptotic cells were observed in close spatial and temporal association with the invasion of peritruncal vessels into the aorta (HH 29–31), the initial formation of coronary orifices (HH 32–33), and the further development of the definitive coronary arteries and orifices (HH 34–35). Whereas the origin of these apoptotic cells and the specific factors regulating their death remain unknown, the results of the present study strongly correlate apoptosis with the formation of proximal coronary arteries and their orifices. Our findings suggest avenues for further research and indicate that factors involved in regulating apoptosis should be included in future models of coronary artery development. Anat Rec 262:310–317, 2001.

Combining topographical and genetic cues to promote neuronal fate specification in stem cells.

There is little remedy for the devastating effects resulting from neuronal loss caused by neural injury or neurodegenerative disease. Reconstruction of damaged neural circuitry with stem cell-derived neurons is a promising approach to repair these defects, but controlling differentiation and guiding synaptic integration with existing neurons remain significant unmet challenges. Biomaterial surfaces can present nanoscale topographical cues that influence neuronal differentiation and process outgrowth. By combining these scaffolds with additional molecular biology strategies, synergistic control over cell fate can be achieved. Here, we review recent progress in promoting neuronal fate using techniques at the interface of biomaterial science and genetic engineering. New data demonstrates that combining nanofiber topography with an induced genetic program enhances neuritogenesis in a synergistic fashion. We propose combining patterned biomaterial surface cues with prescribed genetic programs to achieve neuronal cell fates with the desired sublineage specification, neurochemical profile, targeted integration, and electrophysiological properties.

BDNF profoundly and specifically increases KCNQ4 expression in neurons derived from embryonic stem cells

Neurons resembling the spiral ganglion neurons (SGNs) of the auditory nerve can be generated from embryonic stem cells through induced overexpression of the transcription factor Neurogenin-1 (Neurog1). While recapitulating this developmental pathway produces glutamatergic, bipolar neurons reminiscent of SGNs, these neurons are functionally immature, being characterized by a depolarized resting potential and limited excitability. We explored the effects of two neurotrophins known to be present in the inner ear, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), on the electrophysiology of neurons following Neurog1 induction. Our data reveal a significant reduction in resting membrane potential (RMP) following neurotrophin exposure, with BDNF producing a more robust effect than NT-3. This effect was accompanied by a profound and specific upregulation of the KCNQ4 subtype, where a 9-fold increase was observed with quantitative PCR. The other neuronally expressed KCNQ subtypes (2, 3, and 5) exhibited upregulation which was 3-fold or less in magnitude. Quantitative immunohistochemistry confirmed the increase in KCNQ4 expression at the protein level. The present data show a novel link between BDNF and KCNQ4 expression, yielding insight into the restricted expression pattern of a channel known to play special roles in setting the resting potential of auditory cells and in the etiology of progressive high-frequency hearing loss.

Expression of Neurogenin 1 in Mouse Embryonic Stem Cells Directs the Differentiation of Neuronal Precursors and Identifies Unique Patterns of Down‐stream Gene Expression

Background: Delineating the cascades of growth and transcription factor expression that shape the developing nervous system will improve our understanding of its molecular histogenesis and suggest strategies for cell replacement therapies. In the current investigation, we examined the ability of the proneural gene, Neurogenin1 (Neurog1; also Ngn1, Neurod3), to drive differentiation of pluripotent embryonic stem cells (ESC). Results: Transient expression of Neurog1 in ESC was sufficient to initiate neuronal differentiation, and produced neuronal subtypes reflecting its expression pattern in vivo. To begin to address the molecular mechanisms involved, we used microarray analysis to identify potential down‐stream targets of Neurog1 expressed at sequential stages of neuronal differentiation. Conclusions: ESC expressing Neurogenin1 begin to withdraw from cycle and form precursors that differentiate exclusively into neurons. This work identifies unique patterns of gene expression following expression of Neurog1, including genes and signaling pathways involved in process outgrowth and cell migration, regional differentiation of the nervous system, and cell cycle. Developmental Dynamics 242:230–253, 2013.

Intraspinal transplantation of neurogenin-expressing stem cells generates spinal cord neural progenitors

Embryonic stem (ES) cells and their derivatives are an important resource for developing novel cellular therapies for disease. Controlling proliferation and lineage selection, however, are essential to circumvent the possibility of tumor formation and facilitate the safe translation of ES-based therapies to man. Expression of appropriate transcription factors is one approach to direct the differentiation of ES cells towards a specific lineage and stop proliferation. Neural differentiation can be initiated in ES cells by expression of Neurogenin1 (Ngn1). In this study we investigate the effects of controlled Ngn1 expression on mouse ES (mES) cell differentiation in vitro and following grafting into the rat spinal cord. In vitro, Ngn1 expression in mES cells leads to rapid and specific neural differentiation, and a concurrent decrease in proliferation. Similarly transplantation of Ngn1-expressing mES cells into the spinal cord lead to in situ differentiation and spinal precursor formation. These data demonstrate that Ngn1 expression in mES cells is sufficient to promote neural differentiation and inhibit proliferation, thus establishing an approach to safely graft ES cells into the spinal cord.

Gene Silencing Using RNA Interference in Embryonic Stem Cells

Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy.

Normal Vulva: Embryology, Anatomy, and Histology

The vulva, which is the external genitalia of the female, collectively consists of the mons pubis, labia majora and minora, clitoris, vestibule, greater (Bartholin) and lesser vestibular glands, and vaginal opening (introitus). This chapter describes the gross and microscopic anatomy of the adult, as well as the embryological derivation of the region’s multiple components.