https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/49782/1/Yamanaka_Cell_131_5.pdf

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

Inositol trisphosphate and calcium signalling

If it were possible to reprogram differentiated human somatic cells into a pluripotent state, patient-specific and disease-specific stem cells could be developed. Previous work generated induced pluripotent stem (iPS) cells capable of germline transmission from murine somatic cells by transd

/pdf/induction-of-pluripotent-stem-cells-from-adult-human-3jc4cxk5x5.pdf

Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.

Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.

http://web.mit.edu/7.31/restricted/pdfs/F05_and_earlier/Nichols.pdf

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.

https://www.research.manchester.ac.uk/portal/files/159714472/journal.pone.0174181.pdf

Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics.

Exogenous transferrin is taken up and localized by the neurulation-stage mouse embryo in vitro.

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function.

/pdf/the-maestro-mro-gene-is-dispensable-for-normal-sexual-4x5km6msz9.pdf

The Maestro (Mro) gene is dispensable for normal sexual development and fertility in mice.

Analysis of neural tube defects in a mouse mutant using whole embryo culture.

SUMMARY Theaimofthis study was todetermine whether open lumbosacral spina bifida results froman abnormality ofneural folding (primary neurulation) or medullary cordcanalisation (secondary neurulation). Homozygous curly tail (ct) mouse embryos were studied as a model systemforhumanneural tubedefects. Therostral endofthespina bifida wasfound tolie atthe level ofsomites 27to32inover90%ofaffected ct/ct embryos. Indian inkmarking experiments using non-mutant embryos showed that theposterior neuroporecloses, andprimary neurulation iscompleted, atthelevel ofsomites 32to34.Since neurulation inmammals progressesin a craniocaudal sequence,without overlap between regions ofprimary andsecondary neurulation, we conclude that spina bifida inct/ct embryos arises initially asadefect ofprimary neurulation. Theposition ofposterior neuroporeclosure inhumanembryos isestimated tolie atthelevel of thefuture second sacral segmentindicating that inhumans, asinthectmouse,lumbosacral spina bifida usually arises as a defect ofposterior neuroporeclosure. Cranial NTD affect females predominantly, whereas lower spinal NTD aremore common inmales, bothinhumansandct mice.We offer an explanation forthis phenomenon based on (a)differences intheeffect of embryonic growth retardation on thelikelihood that an embryowilldevelop either cranial or lower spinal NTD and(b)differences intherateofgrowth anddevelopment ofmaleandfemale embryos atthetimeofneurulation.

F. A. Brook

Papers

Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics.

Exogenous transferrin is taken up and localized by the neurulation-stage mouse embryo in vitro.

The Maestro (Mro) gene is dispensable for normal sexual development and fertility in mice.

Analysis of neural tube defects in a mouse mutant using whole embryo culture.

Doeslumbosacral spinabifida arisebyfailure of neural folding or bydefective canalisation