Alpha7 nicotinic acetylcholine receptors and neural network synaptic transmission in human induced pluripotent stem cell-derived neurons.

The α7 nicotinic acetylcholine receptor has been extensively researched as a target for treatment of cognitive impairment in Alzheimer's disease and schizophrenia. Investigation of the α7 receptor is commonly performed in animals but it is critical to increase the biologically relevance of the model systems to fully capture the physiological role of the α7 receptor in humans. For example most humans, in contrast to animals, express the hybrid gene CHRFAM7A, the product of which modulates α7 receptor activity. In the present study, we used human induced pluripotent stem cell (hiPSC) derived neurons to establish a humanized α7 model. We established a cryobank of neural stem cells (NSCs) that could reproducibly be matured into neurons expressing neuronal markers and CHRNA7 and CHRFAM7A. The neurons responded to NMDA, GABA, and acetylcholine and exhibited synchronized spontaneous calcium oscillations. Gene expression studies and application of a range of α7 positive allosteric modulators (PNU-120595, TQS, JNJ-39393406 and AF58801) together with the α7 agonist PNU-282987 during measurement of intracellular calcium levels demonstrated the presence of functional α7 receptors in matured hiPSC-derived neuronal cultures. Pharmacological α7 activation also resulted in intracellular signaling as measured by ERK 1/2 phosphorylation and c-Fos protein expression. Moreover, PNU-120596 increased the frequency of the spontaneous calcium oscillations demonstrating implication of α7 receptors in human synaptic networks activity. Overall, we show that hiPSC derived neurons are an advanced in vitro model for studying human α7 receptor pharmacology and the involvement of this receptor in cellular processes as intracellular signaling and synaptic transmission.

Induced pluripotent stem cell - derived neurons for the study of spinocerebellar ataxia type 3.

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study, induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation, expressed comparable levels of several neuronal markers and responded to the neurotransmitters, glutamate/glycine, GABA and acetylcholine. Additionally, all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation, and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity, although not comparable to that of the adult human brain. Interestingly, we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion, we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity, which could potentially be valuable for the study of SCA3 disease mechanisms.

Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.A11.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a CAG-repeat expanding mutation in ATXN3. We generated induced pluripotent stem cells (iPSCs) from a SCA3 patient by electroporation of dermal fibroblasts with episomal plasmids encoding L-MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of genomically integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. This iPSC line could be useful for the investigation of SCA3 disease mechanisms.

Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.B11.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by an expansion of the CAG-repeat in ATXN3. In this study, induced pluripotent stem cells (iPSCs) were generated from SCA3 patient dermal fibroblasts by electroporation with episomal plasmids encoding L-MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. Potentially, this iPSC line could be a useful tool for the investigation of SCA3 disease mechanisms.

New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

T47D breast cancer cells switch from ER/HER to HER/c-Src signaling upon acquiring resistance to the antiestrogen fulvestrant.

In this study, T47D cell lines resistant to the antiestrogen fulvestrant were established and analyzed to explore, whether a switch to HER signaling, as seen in fulvestrant resistant MCF-7 cell lines, is a general resistance mechanism. We find that parental T47D cells depend on ER and HER signaling for growth. Fulvestrant resistant T47D cells have lost ER expression and, although HER2 was over expressed, growth was only partially driven by HER receptors. Instead c-Src was important for resistant growth. Thus, the T47D and MCF-7 model system unravel different resistance mechanisms which may be important for fulvestrant resistant breast cancer patients.