Small molecules facilitate rapid and synchronous iPSC generation.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-ß inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

Neuronal expression of SOX2 is enriched in specific hypothalamic cell groups.

The transcription factor SOX2 has many established roles in neural development but is generally considered to have limited activity in the adult brain. As part of a study of neuronal phenotypes in the adult rodent hypothalamus, we have now used immunohistochemical analysis to investigate the expression of SOX2 in the adult rat and mouse hypothalamus. Our analysis has revealed that SOX2 protein is extensively expressed in cells of the suprachiasmatic nucleus (SCN). Co-localization with the nuclear marker proteins NeuN and MeCP2 confirmed SOX2 expression in mature neurons of the rat SCN, and the functional integrity of these SOX2+ neurons was also confirmed by demonstrating co-localization with light-induced EGR1 protein. In addition to the SCN, we have also revealed a population of SOX2+/(NeuN+/MeCP2+) neurons in the rat periventricular nucleus (PeN). However, in other hypothalamic nuclei such as the supraoptic nucleus (SON) SOX2+ cells were rare. In extra-hypothalamic areas, SOX2+ cells were also scarce although we have confirmed populations of non-neuronal SOX2+ cells in both the rat sub-ventricular zone (SVZ) and sub-granular zone (SGZ) of the hippocampus. In addition, we have identified an extensive, novel population of non-neuronal SOX2+ cells in the rat subfornical organ (SFO). Our findings provide further evidence of 'immature' phenotypes in rodent SCN neurons and, given the extensive expression of SOX2 across these hypothalamic neurons, may identify a common regulatory factor that maintains this unusual neuronal phenotype. Conservation of SCN SOX2 expression in both rat and mouse indicates a functional requirement for this transcription factor that may be integral to the role of these SCN neurons in mediating daily physiological rhythms.

Generation of human induced pluripotent stem (Ips) cells in serum- and feeder-free defined culture and TGF-Β1 regulation of pluripotency.

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-ß1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-ß1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-ß1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.

Induced pluripotent stem cells from goat fibroblasts.

Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo.

Podoplanin and SOX2 expression in esophageal squamous cell carcinoma after neoadjuvant chemo-radiotherapy.

Neoadjuvant chemo-radiotherapy (CRT) followed by surgery are the standard approaches for locally advanced esophageal cancer. However, the overall cure rate is very low. The aim of this preliminary study was to evaluate the expression of podoplanin and SOX2 known as stemness markers for esophageal squamous cell carcinoma (ESCC) and their association with clinical outcome. We obtained a total of 20 specimens from patients with ESCC who underwent neoadjuvant CRT (30-40 Gy; 5-fluorouracil plus cisplatin) followed by surgery. Podoplanin and SOX2 expression was evaluated using immunohistochemistry and the association of their expressions with clinicopathological variables was investigated. Podoplanin and SOX2 staining was detected not only in residual cancer cells, but also in the basal layer of adjacent normal mucosa after neoadjuvant CRT. High expression of podoplanin was correlated with lymph node metastasis, advanced postoperative stage and vascular invasion (P<0.05), while, high expression of SOX2 was correlated with lymphatic, vascular invasion, poor differentiated tumor and incomplete resection (P<0.05). High expression of podoplanin was significantly associated with poor overall survival (P<0.05). In conclusion, the expression levels of podoplanin and SOX2 expression may be useful prognostic markers for ESCC treated with neoadjuvant CRT.

Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition.

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

Supporting cell characteristics in long-deafened aged mouse ears.

Significant sensory hair cell loss leads to irreversible hearing and balance deficits in humans and other mammals. Future therapeutic strategies to repair damaged mammalian auditory epithelium may involve inserting stem cells into the damaged epithelium, inducing non-sensory cells remaining in the epithelium to transdifferentiate into replacement hair cells via gene therapy, or applying growth factors. Little is currently known regarding the status and characteristics of the non-sensory cells that remain in the deafened auditory epithelium, yet this information is integral to the development of therapeutic treatments. A single high-dose injection of the aminoglycoside kanamycin coupled with a single injection of the loop diuretic furosemide was used to kill hair cells in adult mice, and the mice were examined 1 year after the drug insult. Outer hair cells are lost throughout the entire length of the cochlea and less than a third of the inner hair cells remain in the apical turn. Over 20% and 55% of apical organ of Corti support cells and spiral ganglion cells are lost, respectively. We examined the expression of several known support cell markers to investigate for possible support cell dedifferentiation in the damaged ears. The support cell markers investigated included the microtubule protein acetylated tubulin, the transcription factor Sox2, and the Notch signaling ligand Jagged1. Non-sensory epithelial cells remaining in the organ of Corti retain acetylated tubulin, Sox2 and Jagged1 expression, even when the epithelium has a monolayer-like appearance. These results suggest a lack of marked SC dedifferentiation in these aged and badly damaged ears.