Efficient, high-throughput transfection of human embryonic stem cells

INTRODUCTION: Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. METHODS: We investigated the ability of two commercially available electroporation systems, the Nucleofection(® )96-well Shuttle(® )System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. RESULTS: Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection(® )96-well Shuttle(® )System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. CONCLUSIONS: Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC

Epigenetic regulation and transcription factor programming enhances neurogenesis in neural stem cells

In this thesis, we questioned how neuronal and glial phenotypes become specialized. Epigenetic chromatin modifiers and transcription factors were investigated on their roles in programming and maintaining neural lineage restriction. A relatively homogeneous population of cells was generated by deriving immortalizing neural clones from embryonic rat forebrains. Three phenotypes; neuronal, glial and multipotential (GE6, GE2, CTX8), provided contrasting lineages to probe the factors responsible for shaping cell fate. One particular clone, GE6, differentiated into a functional inhibitory like interneuron. Gene expression analysis showed several genes such as Ascl1, Dlx1, Dlx5, may be responsible for the interneuronal phenotype. Epigenetic regulation through histone modifications is believed to be an essential component within the developing nervous system, ultimately affecting cell fate. Testing chromatin signatures on specific neural genes with permissive and repressive histone “marks” shows that chromatin state in undifferentiated precursors correlates with current and predicts downstream gene expression. These results suggest that cell fate may already be predetermined. Furthermore, ChIP sequencing reveals global differences between the representative clones. Extrinsic growth factors, such as BMP2 promotes the neuronal and glial phenotypes in the multipotential cell CTX8. BMP2 asserts its phenotypic response in part by regulating global acetylation enrichment in specific neural gene networks, providing a mechanism to promote and maintain cell fate. Directly altering chromatin marks using a histone deacetylase inhibitor, valproic acid (VPA), globally acetylates the chromatin of CTX8 cells and enhances neurogenesis. VPA treatment was found to also maintain or increase acetylation in specific neuronal genes, such as Ascl1. In addition, several microRNAs thought to play a role in neurogenesis were also epigenetically regulated after VPA treatment. Finally, through the combination of gene expression and epigenetic analyses, direct programming through exogenous expression of Ascl1, Dlx1 and Dlx5 enhanced neurogenesis in CTX8. Gene expression and epigenetic signature mapping provides us with a deeper understanding of how lineage restriction occurs. Learning the programming rules will assist in directing homogeneous populations of neuronal cells to further probe the mechanisms of neurodegenerative diseases.Ph. D.Includes bibliographical referencesby Christopher L. Ricuper

mTOR acts as a pivotal signaling hub for neural crest cells during craniofacial development.

mTOR is a highly conserved serine/threonine protein kinase that is critical for diverse cellular processes in both developmental and physiological settings. mTOR interacts with a set of molecules including Raptor and Rictor to form two distinct functional complexes, namely the mTORC1 and mTORC2. Here, we used novel genetic models to investigate functions of the mTOR pathway for cranial neural crest cells (NCCs), which are a temporary type of cells arising from the ectoderm layer and migrate to the pharyngeal arches participating craniofacial development. mTOR deletion elicited a proliferation deficit and excessive apoptosis of post-migratory NCCs, leading to growth arrest of the facial primordia along with midline orofacial clefts. Furthermore, NCC differentiation was impaired. Thus, NCC derivatives, such as skeletons, vasculatures and neural tissues were either rudimentary or malformed. We further demonstrate that disruption of mTOR caused P53 hyperactivity and cell cycle arrest in cranial NCCs, and lowering P53 activity by one copy reduction attenuated the severity of craniofacial phenotype in NCC-mTOR knockout mice. Remarkably, NCC-Rptor disruption caused a spectrum of defects mirroring that of the NCC-mTOR deletion, whereas NCC-Rictor disruption only caused a mild craniofacial phenotype compared to the mTOR and Rptor conditional knockout models. Altogether, our data demonstrate that mTOR functions mediated by mTORC1 are indispensable for multiple processes of NCC development including proliferation, survival, and differentiation during craniofacial morphogenesis and organogenesis, and P53 hyperactivity in part accounts for the defective craniofacial development in NCC-mTOR knockout mice

The role of inhibition of osteocyte apoptosis in mediating orthodontic tooth movement and periodontal remodeling: a pilot study

Background Orthodontic tooth movement (OTM) has been shown to induce osteocyte apoptosis in alveolar bone shortly after force application. However, how osteocyte apoptosis affects orthodontic tooth movement is unknown. The goal of this study was to assess the effect of inhibition of osteocyte apoptosis on osteoclastogenesis, changes in the alveolar bone density, and the magnitude of OTM using a bisphosphonate analog (IG9402), a drug that affects osteocyte and osteoblast apoptosis but does not affect osteoclasts. Material and methods Two sets of experiments were performed. Experiment 1 was used to specifically evaluate the effect of IG9402 on osteocyte apoptosis in the alveolar bone during 24 h of OTM. For this experiment, twelve mice were divided into two groups: group 1, saline administration + OTM24-h (n=6), and group 2, IG9402 administration + OTM24-h (n=6). The contralateral unloaded sides served as the control. The goal of experiment 2 was to evaluate the role of osteocyte apoptosis on OTM magnitude and osteoclastogenesis 10 days after OTM. Twenty mice were divided into 4 groups: group 1, saline administration without OTM (n=5); group 2, IG9402 administration without OTM (n=5); group 3, saline + OTM10-day (n=6); and group 4, IG9402 + OTM10-day (n=4). For both experiments, tooth movement was achieved using Ultra Light (25g) Sentalloy Closed Coil Springs attached between the first maxillary molar and the central incisor. Linear measurements of tooth movement and alveolar bone density (BVF) were assessed by MicroCT analysis. Cell death (or apoptosis) was assessed by terminal dUTP nick-end labeling (TUNEL) assay, while osteoclast and macrophage formation were assessed by tartrate-resistant acid phosphatase (TRAP) staining and F4/80+ immunostaining. Results We found that IG9402 significantly blocked osteocyte apoptosis in alveolar bone (AB) at 24 h of OTM. At 10 days, IG9402 prevented OTM-induced loss of alveolar bone density and changed the morphology and quality of osteoclasts and macrophages, but did not significantly affect the amount of tooth movement. Conclusion Our study demonstrates that osteocyte apoptosis may play a significant role in osteoclast and macrophage formation during OTM, but does not seem to play a role in the magnitude of orthodontic tooth movement

Lowering P53 activity by <i>P53</i> copy reduction attenuates the craniofacial phenotype in the NCC-<i>mTOR</i> cKO mice.

<p>(A) Gross examination of NCC-<i>mTOR</i> cKO mice by <i>P53</i> copy deduction. (B) Alcian blue staining in parasagittal sections of E12.5 heads. (C) Histology in frontal sections of E12.5 heads. (D) Quantification of the length of snout/frontonasal prominence, which is represented by the length from the brain-nose turning point to the most anterior plane of the snout. (E) PHH3 staining in frontal sections of E11.5 FN. (F) Apoptosis in frontal sections of E11.5 heads (arrow heads). (G, H) Quantification of cell proliferation and apoptosis. * P<0.05; **P<0.01. bc: basicranium; br; brain; ctr: control; fn: frontonasal prominence; ls: length of the snout; mc: Meckel’ cartilage; md: mandibular arch; mx: maxillary arch; ne: nasal epithelium; pl: palatal shelf; sn: snout; tb: tooth bud; ton: tongue; vd: vessel dilation. Scale bar: 200μm.</p

Apoptosis and proliferation assays.

<p>(A-D) Apoptosis at E10.5 and E11.5. (E) Quantification of apoptotic cells. (F, F’) Dual staining of TUNEL and Runx2 shows that apoptotic cells are predominantly NCC descendants. (G, H) Immunofluorescence for PHH3 in E10.5 facial primordia. (I, J) Immunofluorescence for PHH3 in the FN at E11.5. (K, L) Immunofluorescence for PHH3 in the mandibular arch at E11.5. Arrowheads indicate groove in the tongue. (M) Quantification of PHH3+ cells. (N, O) O9 NCCs cultured with and without rapamycin (100nM). (P) PHH3 staining of O9 cells. (Q)Western blot for mTORC1 downstream target p-S6K1/S6K1 upon rapamycin treatment. (R) Percentage of PHH3+ cells and cell live/death assay. (S) Phase contrast images of cultured PA cells. (T) Phalloidin and Dapi staining of PA cells. (U) PHH3 and Phalloidin double staining of PA cells. br: brain; ctr: control; fn: frontonasal prominence; md: mandibular prominence; mx: maxillary prominence; rapa: rapamycin; ton: tongue. Scale bars in (A-D): 100 μm.; scale bars in (L-U): 200 μm; scale bar in (L) applies to (G-K).</p

A positive feedback mechanism that regulates expression of miR-9 during neurogenesis.

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism

mTOR dynamics during craniofacial development.

<p>(A) Whole mount LacZ staining of <i>Wnt1-cre; Rosa26R</i> embryos. White lines indicate the sectioning planes in relevant images. (B) Immunofluorescence for Ap2-α and p-mTOR in the migrating NCCs at E9.0. (C, D; F, G) Immunofluorescence for p-mTOR in the FN and mandibular prominences at late E10.5. (E, H) Sections of a Lac-Z-stained E10.5 heads, showing NCC distribution in the FN and mandibular prominence. (I, J, K) Co-localization of p-mTOR with a neurofilament marker 3A10. (L, M) Immunofluorescence for p-mTOR in the mandibular prominence of E10.5 mice. (N) Quantification of p-mTOR levels of the mandibular prominences by calculating relative fluorescence intensity, **p<0.01. br: brain; ctr: control; fn: frontonasal prominence; cko: conditional knockout; md: mandibular prominence; md.b: mandibular branch of trigeminal nerve; mx: maxillary prominence; ne: nasal epithelium; nt: neural tube; tg: trigeminal ganglion; ton: tongue. Scale bar in (A): 200 μm; Scale bars in others: 100 μm.</p

Defective craniofacial morphogenesis and organogenesis in the NCC-<i>mTOR</i> KO mice.

<p>(A) Craniofacial morphogenesis at two successive stages. Arrowheads point to facial cleft. (B) Lateral view of E11.5 heads, NCCs are GFP-labeled. (C) SEM examination of E12.5 heads, * marks facial cleft. (D, E) Histology of E11.5 heads, showing failed midline fusion of the mutant FNs. (F, G) Histology of E14.5 heads. (H-K) Immunofluorescence for β-catenin and p-Smad1/5/8, arrowheads point to positive staining in the mandibular prominences. (L) Quantification of β-catenin and p-Smad1/5/8 levels by calculating relative fluorescence intensity. (M-Q) Whole mount in situ hybridization for <i>Alx3</i>, <i>Msx1</i>, <i>Pax3</i> and <i>Fgf8</i>. (R) Quantification of in situ hybridization staining of <i>Alx3</i>, <i>Msx1</i>, <i>Pax3</i> and <i>Fgf8</i>. *P<0.05; ** P<0.01. br: brain; ctr: control; fn: frontonasal prominence; md: mandibular prominence; mx: maxillary prominence; ns: non-significant; pal: palate; sn: snout; tb: tooth bud; ton: tongue; vs: vessel. Scale bar in (A-C): 100 μm; scale bar in (A) applies to (M-Q); scale bars in others: 200 μm.</p