Long term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1-ROCK-Myosin II and TGF-β signaling

Summary: Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine. : Zhang et al. screen a small-molecule collection and find that pharmacologic inhibition of TGF-β and PAK1-ROCK-Myosin II, in low calcium conditions, supports extended expansion of epithelial stem cells in 2D format. This approach enhances the potential of tissue-resident epithelial stem cells for cell therapy. Keywords: epithelial stem and progenitor cells, cell culture method, TGF-β, PAK1/ROCK/Myosin II, feeder-free, regenerative medicine, cell therap

Neuromodulation of the feedforward dentate gyrus-CA3 microcircuit

The feedforward dentate gyrus-CA3 microcircuit in the hippocampus is thought to activate ensembles of CA3 pyramidal cells and interneurons to encode and retrieve episodic memories. The creation of these CA3 ensembles depends on neuromodulatory input and synaptic plasticity within this microcircuit. Here we review the mechanisms by which the neuromodulators aceylcholine, noradrenaline, dopamine, and serotonin reconfigure this microcircuit and thereby infer the net effect of these modulators on the processes of episodic memory encoding and retrieval

Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages

Generation of stable PDX derived cell lines using conditional reprogramming

Abstract Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts

The role of transcriptional ‘futile cycles’ in autophagy and microbial pathogenesis

Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, where by the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional ‘futile cycle’ where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novosynthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformansresults in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a keyrole in the facilitation of microbial pathogenesis

The role of transcriptional ‘futile cycles’ in autophagy and microbial pathogenesis

Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, where by the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional ‘futile cycle’ where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novosynthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformansresults in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a keyrole in the facilitation of microbial pathogenesis

PTX treatment does not affect the autophagic activity in the human macrophage cell line THP-1.

<p>(<i>A</i>) Immunoblot analysis of THP-1 cell lysates untreated; or pre-exposed to PTX (200 ng/ml for 2h), either not further manipulated, starved (HBSS for 2h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 2h) to assess LC3-II/actin band ratios. (<i>B</i>) Fluorescent microscopic images of LC3-GFP expression in a THP-1 GFP-LC3 stable cell line pre-exposed to PTX, or not and starved (HBSS for 2h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 2h). Representative images are shown for each condition. (<i>C</i>) Quantification of GFP-LC3 dots in 50-100 cells for each condition from experiment shown in part B. Data represents the mean LC3 puncta per cytosol ± SEM from four independent experiments.</p

The lack of Gα_i3 or PTX treatment does not affect autophagy induction in primary mouse macrophages.

<p>(<i>A</i>) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3^-/- mice; or wild type mice pre-exposed to PTX (200 ng/ml for 2h), or not, to assess the steady state levels of Gα_i3, Gα_i2, p62 and ubiquitin proteins. (<i>B</i>) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3^-/- mice; or wild type mice pre-exposed to PTX as above, or not, either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios. (<i>C</i>) Endogenous LC3 immunocytochemistry of BMDM from wild type mice, Gnai3^-/- mice, or from wild type mice pre-exposed to PTX (200 ng/ml for 2h) either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h). Representative images are shown for each condition. (<i>D</i>) Quantification of endogenous LC3 dots performed by fluorescence microscopy for at least 70-100 cells from experiment shown in part C. Data represents the mean LC3 puncta per cytosol ± SEM for three independent experiments for each condition. (<i>E</i>) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7^-/- mice to assess the steady state levels of p62 and ubiquitin proteins. (<i>F</i>) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7^-/- deficient mice, either not further manipulated, or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios.</p

PTX treatment, the lack of Gα_i3, or the lack of AGS3 does not affect the anti-autophagic activity in primary mouse macrophages.

<p>(<i>A</i>) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; or wild type mice pre-exposed to PTX (<i>B</i>) Gnai3^-/- mice; (<i>C</i>) Gpsm1^-/- mice, either not further manipulated, treated with rapamycin (50 μg/ml for 2h), treated with complete media alone for 2-3 hours following rapamycin (50 μg/ml for 2h) treatment. (<i>D</i>) Endogenous LC3 immunocytochemistry of BMDM from wild type mice, wild type mice pre-exposed to PTX (200 ng/ml for 2h), Gnai3^-/- mice, and Gpsm1^-/- mice either not further manipulated, treated with rapamycin (50 μg/ml for 2h), or treated with complete media alone for 2-3 hours following rapamycin (50 μg/ml for 2h) treatment. Representative images are shown for each condition. </p