A simple and efficient system for regulating gene expression in human pluripotent stem cells and derivatives.

Regulatable transgene expression in human pluripotent stem cells (hPSCs) and their progenies is often necessary to dissect gene function in a temporal and spatial manner. However, hPSC lines with inducible transgene expression, especially in differentiated progenies, have not been established due to silencing of randomly inserted genes during stem cell expansion and/or differentiation. Here, we report the use of transcription activator-like effector nucleases-mediated targeting to AAVS1 site to generate versatile conditional hPSC lines. Transgene (both green fluorescent protein and a functional gene) expression in hPSCs and their derivatives was not only sustained but also tightly regulated in response to doxycycline both in vitro and in vivo. We modified the donor construct so that any gene of interest can be readily inserted to produce hPSC lines with conditional transgene expression. This technology will substantially improve the way we study human stem cells.

Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.

Regulation of myeloid ecotropic viral integration site 1 and its expression in normal and abnormal endometrium.

OBJECTIVE: To identify the expression profile and sex steroid regulation pattern of myeloid ecotropic viral integration site 1 (MEIS1) in endometrium. DESIGN: Molecular studies in human and animal tissue. SETTING: Reproductive medicine center of a university hospital. PATIENT(S) AND ANIMAL(S): Women with normal menstrual cycles for male infertility and female infertility with endometriosis. Sexually mature female mice (Kunming White strain). INTERVENTION(S): Primary cultured endometrial stromal cells, Ishikawa cells, and oophorectomized mice were treated with sex steroid. MAIN OUTCOME MEASURE(S): MEIS1 expression in the human endometrium during the menstrual cycle, mouse uterus during the peri-implantation period of pregnancy, and eutopic endometrium from patients with endometriosis was analyzed by immunohistochemistry staining and western blot. In addition, MEIS1 expression in response to sex steroid was examined both in vitro and in vivo by immunohistochemistry staining and western blot. RESULT(S): MEIS1 expression was markedly increased in endometrium during the implantation period, and in decidualizing stromal cells in human endometrium and murine uterus. Steroid hormones increased MEIS1 expression in primary cultured endometrial stromal cells, Ishikawa cells, and endometrium of oophorectomized mice. The effects of estrogen and progesterone were more marked in oophorectomized mice and were additive. MEIS1 expression was significantly lower in eutopic endometrium compared with normal endometrium in the midsecretory stage. CONCLUSION(S): MEIS1 is likely a key mediator between sex steroid and genes for uterine receptivity. Diminished endometrium MEIS1 expression may contribute to implantation failure in endometriosis.

Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons.

Amyotrophic lateral sclerosis (ALS) presents motoneuron (MN)-selective protein inclusions and axonal degeneration but the underlying mechanisms of such are unknown. Using induced pluripotent cells (iPSCs) from patients with mutation in the Cu/Zn superoxide dismutase (SOD1) gene, we show that spinal MNs, but rarely non-MNs, exhibited neurofilament (NF) aggregation followed by neurite degeneration when glia were not present. These changes were associated with decreased stability of NF-L mRNA and binding of its 3' UTR by mutant SOD1 and thus altered protein proportion of NF subunits. Such MN-selective changes were mimicked by expression of a single copy of the mutant SOD1 in human embryonic stem cells and were prevented by genetic correction of the SOD1 mutation in patient's iPSCs. Importantly, conditional expression of NF-L in the SOD1 iPSC-derived MNs corrected the NF subunit proportion, mitigating NF aggregation and neurite degeneration. Thus, NF misregulation underlies mutant SOD1-mediated NF aggregation and axonal degeneration in ALS MNs.