Name: de novo generation of somatic stem cells by yap/taz
Uploaded: 2018-05-07T14:00:00.000+00:00
Duration: 13 min 5 s
Description: Watch this Scientific Journal Video about De Novo Generation of Somatic Stem Cells by YAP/TAZ at JoVE.com

We thank F. Camargo for the gift of tetO- YAPS127A mice; R26-rtTAM2 mice (stock #006965) were purchased from The Jackson Laboratory. We thank Chiara Frasson and Giuseppe Basso for help with FACS procedures. This work is supported by AIRC Special Program Molecular Clinical Oncology &#39;&#39;5 per mille&#39;&#39; and an AIRC PI-Grant to S.P, and by Epigenetics Flagship project CNR-Miur grants to S.P. This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation programme (grant agreement DENOVOSTEM No. 670126).

<ol>
	<li>Panciera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Expandable+Tissue-Specific+Stem/Progenitor+Cells+through+Transient+Expression+of+YAP/TAZ.">Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.</a> Cell Stem Cell. 19 (6), 725-737 (2016).</li><li>Bar-Nur, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+conversion+induced+by+pluripotency+factors+involves+transient+passage+through+an+iPSC+stage.">Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage.</a> Nat Biotechnol. 33 (7), 761-768 (2015).</li><li>Xu, J., Du, Y., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+reprogramming:+strategies,+mechanisms,+and+applications.">Direct lineage reprogramming: strategies, mechanisms, and applications.</a> Cell Stem Cell. 16 (2), 119-134 (2015).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Development+and+Disease+with+Organoids.">Modeling Development and Disease with Organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Blanpain, C., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+plasticity.+Plasticity+of+epithelial+stem+cells+in+tissue+regeneration.">Stem cell plasticity. Plasticity of epithelial stem cells in tissue regeneration.</a> Science. 344 (6189), 1242281 (2014).</li><li>Bai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yes-associated+protein+regulates+the+hepatic+response+after+bile+duct+ligation.">Yes-associated protein regulates the hepatic response after bile duct ligation.</a> Hepatology. 56 (3), 1097-1107 (2012).</li><li>Cai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+signaling+pathway+restricts+the+oncogenic+potential+of+an+intestinal+regeneration+program.">The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program.</a> Genes Dev. 24 (21), 2383-2388 (2010).</li><li>Elbediwy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+signalling+regulates+YAP+and+TAZ+to+control+skin+homeostasis.">Integrin signalling regulates YAP and TAZ to control skin homeostasis.</a> Development. 143 (10), 1674-1687 (2016).</li><li>Taniguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gp130-Src-YAP+module+links+inflammation+to+epithelial+regeneration.">A gp130-Src-YAP module links inflammation to epithelial regeneration.</a> Nature. 519 (7541), 57-62 (2015).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downstream+of+mutant+KRAS,+the+transcription+regulator+YAP+is+essential+for+neoplastic+progression+to+pancreatic+ductal+adenocarcinoma.">Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma.</a> Sci Signal. 7 (324), ra42 (2014).</li><li>Zanconato, F., Cordenonsi, M., Piccolo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP/TAZ+at+the+Roots+of+Cancer.">YAP/TAZ at the Roots of Cancer.</a> Cancer Cell. 29 (6), 783-803 (2016).</li><li>Azzolin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP/TAZ+incorporation+in+the+beta-catenin+destruction+complex+orchestrates+the+Wnt+response.">YAP/TAZ incorporation in the beta-catenin destruction complex orchestrates the Wnt response.</a> Cell. 158 (1), 157-170 (2014).</li><li>Stingl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+unique+properties+of+mammary+epithelial+stem+cells.">Purification and unique properties of mammary epithelial stem cells.</a> Nature. 439 (7079), 993-997 (2006).</li><li>Means, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+epithelial+plasticity+mediated+by+acinar+cell+transdifferentiation+and+generation+of+nestin-positive+intermediates.">Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates.</a> Development. 132 (16), 3767-3776 (2005).</li><li>Shi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+acinar+cell+organization+is+critical+to+preventing+Kras-induced+acinar-ductal+metaplasia.">Maintenance of acinar cell organization is critical to preventing Kras-induced acinar-ductal metaplasia.</a> Oncogene. 32 (15), 1950-1958 (2013).</li><li>Huch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> EMBO J. 32, 2708-2721 (2013).</li><li>Fernandez Vallone, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trop2+marks+transient+gastric+fetal+epithelium+and+adult+regenerating+cells+after+epithelial+damage.">Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.</a> Development. 143 (9), 1452-1463 (2016).</li><li>Yanger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+cellular+reprogramming+occurs+spontaneously+during+liver+regeneration.">Robust cellular reprogramming occurs spontaneously during liver regeneration.</a> Genes Dev. 27 (7), 719-724 (2013).</li><li>Yui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP/TAZ-Dependent+Reprogramming+of+Colonic+Epithelium+Links+ECM+Remodeling+to+Tissue+Regeneration.">YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration.</a> Cell Stem Cell. , (2017).</li><li>Pan, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+patterns+of+multipotentiality+in+Ptf1a-expressing+cells+during+pancreas+organogenesis+and+injury-induced+facultative+restoration.">Spatiotemporal patterns of multipotentiality in Ptf1a-expressing cells during pancreas organogenesis and injury-induced facultative restoration.</a> Development. 140 (4), 751-764 (2013).</li><li>Panciera, T., Azzolin, L., Cordenonsi, M., Piccolo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+YAP+and+TAZ+in+physiology+and+disease.">Mechanobiology of YAP and TAZ in physiology and disease.</a> Nat Rev Mol Cell Biol. 18 (12), 758-770 (2017).</li></ol>

The authors declare no competing financial interests.

Here we present protocols to reprogram ex vivo terminally differentiated epithelial cells of different tissues into their corresponding tissue-specific progenitor cells (or ySCs) by transient expression of YAP, as reported previously1. We have detailed two procedures: one allowing reprogramming of FACS-purified cells through lentiviral vectors and a second one that avoids viral infection and takes advantage of transgenic YAP expression. Each protocol presents an efficient strategy to isolate and culture primary differentiated cells and a strategy to force exogenous YAP gene expression in differentiated cells, generating de novo somatic tissue-specific expandable stem cells (see schemes in Figures 1A and 2A).
We demonstrated that the isolation strategies here presented effectively isolate a pure population of differentiated cells, as demonstrated by the fact that we never detected any outgrowth from negative control samples (Figures 1C and 2C).
The lentiviral vectors used in this study for the reprogramming of primary mammary LD cells are doxycycline inducible, offering the possibility of a tight control of the transgene expression; this allows to turn on and off exogenous YAP expression at will. Particular attention should be placed in avoiding the use of an excessive viral titer, as this can be detrimental in terms of reprogramming efficiency. In the case of primary acinar cells, we switched to a fully transgenic approach to obtain a YAP-dependent reprogramming with minimal manipulations. This latter strategy is also particularly appropriate for primary pancreatic acini, as isolated acinar clusters are scarcely amenable to lentiviral infection and very fragile. The transgenic strategy employed offers the same advantage of doxycycline-dependent lentiviral vectors for the tight control of gene expression. Moreover, the transgenic strategy exploited with primary pancreatic acini bears the additional advantage of a much higher reprogramming efficiency compared to the viral-induced reprogramming of mammary LD cells. Beyond the different intrinsic plasticity associated to cells derived from different tissues, the higher rate of pancreatic reprogramming might be derived from the higher efficiency of expression associated to uniform and autonomous YAP expression in all explanted cells. Notably, we have demonstrated that exogenous YAP is no longer required after generation of ySCs (yMaSC colonies and yDucts), without affecting their self-renewal capacity. This is because ySCs reactivate endogenous YAP/TAZ and use them for self-renewal when exogenous YAP is turned off1.
We validated the notion that ySCs indeed emerge from differentiated cells by controlling the cell of origin of our reprogramming experiments through genetic lineage-tracing validations1.
Extensive characterization of ySCs shows that YAP-induced reprogramming generates normal somatic SCs1 as i) at the transcriptomic level, ySCs display massive overlaps with native SCs; ii) ySCs display differentiation potential and can generate a multilineage progeny always restricted to the identity of their tissue of origin; iii) ySCs are non-transformed and non-tumorigenic when transplanted in vivo.
Here we also describe procedures to maintain and expand in culture both yMaSCs and yDucts as organoids embedded in 100% basement membrane matrix hydrogels. These conditions allow for the self-organization of ySCs into three-dimensional organoids that ensure the maintenance of stemness properties long-term in culture, enabling to expand these stem populations at will for downstream analyses and applications. For unknown reasons, we failed to obtain yMaSC organoids by placing infected LD cells directly in organoid culture conditions 7 days after doxycycline treatment in plastic tissue culture plate; in other words, the intermediate growth step in mammary colony conditions is essential. In our hands, even native MaSCs require mammary colony conditions before passaging in organoid culture. Furthermore, the most efficient organoid outgrowth is obtained when we avoid dissociating the primary colonies into single cells, but rather transfer the intact colonies into organoid culture conditions.
Organoid culture conditions also bear the advantage of giving the possibility to cryopreserve ySCs, provided that the organoids are recovered from their matrices, avoiding cell dissociation prior to cryopreservation in nitrogen bath.
The YAP reprogramming procedure presented can convert distinct differentiated cell types derived from different adult tissues into their corresponding tissue-specific stem cells (we have tested it using mammary, pancreatic and neuronal cells)1. At difference from iPSCs or other reprogramming efforts, YAP/induced SCs can maintain the memories of their tissue of origin. Of note, de-differentiation of somatic cells into cells endowed with stem-like properties is the only form of cell fate plasticity and reprogramming observed in vivo, for example after tissue damage and to support wound healing5,17,18,19,20. It is noteworthy that YAP and TAZ are largely dispensable for normal homeostasis but crucial for tissue repair in multiple tissues11,21. Consistently with a physiological function of the reprogramming steps here described, YAP/TAZ have been recently shown to be required in intestinal regeneration in mouse models of ulcerative colitis patients by causing a conversion of adult intestinal cells into a repairing epithelium that displays features of the fetal gut19. YAP reprogramming thus expands the current induced cell plasticity strategies by providing a means to generate somatic stem cells, a state that has been so far challenging to capture in vitro. This approach, if extended also to human-derived cells, might have broad relevance from regenerative medicine applications to the study of the somatic stemness state and for expansion of somatic stem cells in vitro.

Tissue-specific somatic stem cells (SCs) are critical for tissue renewal and repair after injury. The possibility to easily isolate and unlimitedly expand ex vivo somatic SCs represents a critical issue for potential regenerative therapies, as well as for SC applications in basic research and disease modeling. Progress in this direction, however, has been limited by the difficulty of capturing the SC state of various epithelial organs in vitro. Indeed, in several adult tissues resident SCs may not exist, or not be readily available, or their number and regenerative potential may be eroded by aging or disease conditions. In 2016, we started to fill this gap by reporting that expression of a single transcriptional coactivator, YAP (YES-associated protein) or its closely related protein TAZ (transcriptional activator with a PDZ motif), into terminally differentiated cells efficiently creates functional, expandable, non-tumorigenic, autologous cell populations that are operationally and molecularly indistinguishable from their corresponding tissue-specific SCs1. A pulse of sustained YAP or TAZ activity for few days is sufficient to induce the appearance of self-renewing somatic SCs. This is a stable condition that is no longer dependent on continuous transgene expression, as it can be transmitted through cell generations without further expression of ectopic YAP/TAZ1. The protocol presented here details the procedure used to generate de novo epithelial stem/progenitor cells of the mammary gland and pancreas, starting from differentiated cells of these tissues. This procedure fills a black box in the current reprogramming/transdifferentiation arena. Main efforts in these directions have indeed so far centered on cell transition to an induced pluripotent stem cell (iPSC) state, followed by conversion of these embryonic and pluripotent SCs into more differentiated cells. However, iPSCs are tumorigenic once introduced in adult tissues, raising the need of developing protocols for their complete and efficient differentiation2. However, this differentiation step, even when possible, comes at the price of long-term expandability, self-organization and organ repopulation potentials. These are essential attributes for organ regeneration that are in fact typical only of endogenous tissue-specific SCs and of the presently described YAP-induced SCs (ySCs). Similarly, direct transdifferentiation of one cell type into another by using cocktails of various transcription factors also generate differentiated cells that lack essential proliferative and stemness potential3.
The procedure described here also takes advantage of the recently introduced organoid technology, by which endogenous SCs can be expanded and differentiated ex vivo4. YAP-induced SCs may generate organoid-forming SCs even in experimental, biological or disease conditions in which endogenous SCs are not present. We would like to note that, at the difference with other reprogramming procedures, the type of cell plasticity imparted by YAP may correspond to the only form of the reversion to a SC-like status that occurs in living tissues. Reacquisition of SC-like traits has been associated with tissue repair or oncogenic activation5. Although dispensable for the homeostasis of several adult tissues, YAP and/or TAZ are absolutely essential for regeneration, tumor growth and expansion of somatic SCs in vitro1,6,7,8,9,10,11,12

<table><tbody><tr><td>10 mL sterile syringes</td><td>Rays</td><td>10LC</td><td></td></tr><tr><td>100 mm&amp;nbsp; cell strainer</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>15 mL sterile conical tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>24-well ultra low attachment plates</td><td>Costar</td><td>3473</td><td></td></tr><tr><td>40 mm cell strainers</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>48-well multiwell plates</td><td>Corning</td><td>353078</td><td></td></tr><tr><td>50 mL sterile conical tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>6-well multiwell plates</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634028</td><td></td></tr><tr><td>B27 supplement (50x)</td><td>Gibco</td><td>17504001</td><td></td></tr><tr><td>BPE</td><td>Gibco</td><td>13028014</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9418</td><td></td></tr><tr><td>Collagenase, type I</td><td>Sigma</td><td>17018029</td><td></td></tr><tr><td>dexamethasone</td><td>Sigma</td><td>D4902&amp;nbsp;</td><td></td></tr><tr><td>Dispase</td><td>Gibco</td><td>1705-041</td><td></td></tr><tr><td>Disposable scalpels</td><td>Swann-Morton</td><td>0503</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>DnaseI</td><td>Roche</td><td>11284932001</td><td></td></tr><tr><td>doxycycline hyclate</td><td>Sigma</td><td>D9891&amp;nbsp;</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>Ethanol 100%</td><td>Sigma</td><td>51976</td><td></td></tr><tr><td>FACS tubes (with strainer caps)</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>FITC anti-mouse CD326 (Ep-CAM)</td><td>BioLegend</td><td>118208</td><td></td></tr><tr><td>FUdeltaGW-rtTA&amp;nbsp;</td><td>Addgene</td><td>&amp;nbsp;#19780&amp;nbsp;</td><td></td></tr><tr><td>FUW-tetO-EGFP</td><td>Addgene</td><td>&amp;nbsp;#84041</td><td>used as negative control</td></tr><tr><td>FUW-tetO-MCS</td><td>Addgene</td><td>&amp;nbsp;#84008</td><td>used as negative control</td></tr><tr><td>FUW-tetO-wtYAP</td><td>Addgene</td><td>&amp;nbsp;#84009</td><td></td></tr><tr><td>FUW-tetO-YAPS94A</td><td>Addgene</td><td>&amp;nbsp;#84010</td><td>used as negative control (transcriptionally dead YAP mutant)</td></tr><tr><td>GlutaMax</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>24020117</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>30721</td><td></td></tr><tr><td>heparin sodium salt</td><td>Sigma</td><td>H3149&amp;nbsp;</td><td></td></tr><tr><td>HEPES buffer solution (1M)</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>human R-Spondin1 (His Tag)</td><td>Sino Biological</td><td>11083-H08H-5</td><td></td></tr><tr><td>Hyaluronidase from bovine testes</td><td>Sigma</td><td>H3506&amp;nbsp;</td><td></td></tr><tr><td>ITS-X</td><td>Gibco</td><td>51500056</td><td></td></tr><tr><td>K-14 antibody</td><td>Life Technologies</td><td>Ab7800&amp;nbsp;</td><td></td></tr><tr><td>K-8 antibody</td><td>Life Technologies</td><td>Ab14053&amp;nbsp;</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>&amp;nbsp;25030081</td><td></td></tr><tr><td>Lin (allophycocyanin [APC] mouse lineage antibody cocktail)</td><td>BD Biosciences</td><td>51-9003632</td><td></td></tr><tr><td>Matrigel&amp;reg; 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de novo generation of somatic stem cells by yap/taz

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. 
The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

Generation of yMaSCs 
An overview of the experimental strategy to reprogram primary mammary LD cells by transient expression of YAP is presented in Figure 1A. Primary mammary LD epithelial cells are purified by fluorescent-activated cell sorting13. Figure 1B represents a typical sorting procedure to obtain three distinct subpopulations: Basal cells (EpCAMlowCD49fhighCD61-), Luminal Progenitor (LP) cells (EpCAMhighCD49flowCD61+) and LD Cells (EpCAMhighCD49flowCD61-). Careful gating of the three subpopulations is essential to isolate a pure preparation of LD cells, that are fully differentiated and completely growth arrested when seeded in mammary gland colony forming conditions (see Figure 1C, left panel). Conversely, when induced to express exogenous YAP, LD cells start proliferating to form easily recognizable dense epithelial colonies in 5% basement membrane matrix suspension cultures (Figure 1C). The efficiency of reprogramming, attested around 3% for a typical experiment, can be scored by counting the number of colonies over the number of single cells originally seeded in basement membrane matrix suspension cultures (Figure 1D). Reprogrammed luminal cells (yMaSCs) can then be passage into 100% basement membrane matrix organoid culture conditions (see scheme in Figure 1A), self-organizing into complex organoid-like structures that develop around multiple lumens and display remarkable self-renewal ability even in absence of doxycycline (i.e. in absence of transgenic YAP expression) (Figure 1E). Histologically, yMaSC-derived organoids display a basal layer (K14 positive), facing the basement membrane matrix reconstituted ECM and a luminal layer (K8 positive), facing the lumen-like cavities within the organoid (Figure 1F). This architecture is indistinguishable from that of organoids formed by native MaSCs (Figure 1F).
Generation of yDucts 
An overview of the experimental strategy to reprogram primary pancreatic acini by transient expression of YAP is presented in Figure 2A. Entire acinar clusters are isolated from the bulk of the pancreatic tissue by a combination of mild dissociation and size exclusion through filtration. A typical preparation is presented in Figure 2B. After isolation, the acinar cell clusters should appear as a suspension of exocrine acinar units of homogeneous size, with no contamination by endocrine Langerhans islets or fragments of the pancreatic ductal tree and minimal dissociation to single cells. Contamination by endocrine islets or ductal fragments is an indication of deficient selective filtration (step 2.1.10), possibly due to harsh handling; unwanted dissociation of acinar clusters to single cells might be due to excessive collagenase treatment or unbuffered activity of proteolytic enzymes released by the tissue, which can be curbed by additional SBTI treatment.
A typical acinar reprogramming experiment is presented in Figure 2C; within 5-7 days of culture in 3D Collagen-I based hydrogel in the presence of doxycycline, pancreatic acini derived from R26-rtTAM2/tetO-YAPS127A mice readily turn into duct-like clusters (that we named yDucts), composed by a thin monolayer of epithelial cells that proliferate around an expanding central cavity. The reprogramming efficiency, which is around 70% for a typical experiment, can be easily measured by scoring the number of duct-like clusters over the total number of seeded acini (Figure 2D). The negative control cells, that is R26-rtTAM2/+ cells or R26-rtTAM2/tetO-YAPS127A cells left without doxycycline, invariably remain as post-mitotic acinar clusters in these culture conditions, as previously reported1,14,15. Reprogrammed yDucts can then be passaged at the single cell level into Matrigel-based organoid culture conditions16 (see scheme in Figure 2A), displaying remarkable self-renewal ability even in absence of doxycycline (i.e. in absence of transgenic YAP expression) (Figure 2E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57462/57462fig1.jpg" /> 
Figure 1: Isolation of primary mammary LD cells and induction of mammary stem cells. (A) Schematic representation of the experimental procedure adopted to reprogram primary mammary LD cells. (B) Representative FACS-plots illustrating a typical sorting procedure to purify LD cells. i) dissociated cells are gated according to forward and side scatter for live cells (P1; blue); ii) population P1 is then further gated according to its Lin profile: the subpopulation of Lineage-negative cells (P2; grey) is selected, excluding Lineage-positive hematopoietic cells; iii) population P2 is then separated into an EpCAMhigh (P3; yellow + green) and an EpCAMlow (P6; red) subpopulations; iv) P3 and P6 are then further gated according to their CD61/CD49f profile into three subpopulations: EpCAMlowCD49fhighCD61- Basal cells (P7; red), EpCAMhighCD49flowCD61+ LP cells (P8; yellow) and EpCAMhighCD49flowCD61- LD cells (P9; green). (C) Images are illustrative of the ability of LD cells, infected with the indicated constructs, to form mammary colonies 15 days after seeding in mammary colony medium. Only YAP-expressing cells turn into colony-forming cells, whereas negative control cells (EGFP-infected) remain as growth-arrested single cells. Scale bar = 50 &#956;m. (D) Quantification of the colony forming ability of the indicated cells, as in (C). Data are presented as mean + s.d. and are representative of five independent experiments, each with six technical replicates. (E) Representative image of YAP-reprogrammed mammary stem cell-like cells (yMaSCs) after 12 days into organoid culture conditions in fresh three-dimensional 100% basement membrane matrix hydrogel in the absence of Doxycycline. Scale bar = 100 &#956;m. (F) Representative immunofluorescence images for the basal marker K14 (green) and the luminal marker K8 (red) of organoids derived from the indicated cells, after 12 days into organoid culture conditions. Scale bar = 10 &#956;m. This figure is reproduced from Panciera et al., 20161. <a href="/files/ftp_upload/57462/57462fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57462/57462fig2.jpg" /> 
Figure 2: Isolation of primary pancreatic acinar cells and induction of pancreatic progenitors. (A) Schematic representation of the experimental procedure adopted to reprogram primary pancreatic exocrine acinar cells. (B) Representative image of primary pancreatic acini just after the isolation procedure (step 2.1.14). The acinar preparation should appear as a homogeneous suspension of acinar clusters, with minimal presence of single cells. Scale bar = 400 &#956;m. (C) Representative images of primary pancreatic acini derived from R26-rtTAM2 (upper panels)or R26-rtTAM2; tetO-YAPS127A (lower panels) mice and cultured in 3-D Collagen I -based hydrogel for 5 days with or without Doxycycline (doxy), as indicated. Only YAP-expressing primary acini convert to cells growing as cyst-like organoid after Doxycycline addition. Scale bars = 70 &#956;m. (D) Quantification of the ability of pancreatic acini to form ductal organoids upon transgenic YAP overexpression as in (C). Data are presented as mean + s.d. and are representative of five independent experiments, performed with four technical replicates. (E) Representative image of YAP-reprogramed ductal-like cells (yDucts) after three passages in fresh three-dimensional 100% basement membrane matrix hydrogel in the absence of Doxycycline. Scale bar = 130 &#956;m. <a href="/files/ftp_upload/57462/57462fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Isolation of primary mammary cells</td>
		</tr>
		<tr>
			<td>Ca2+ chelating solution</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0.02% w/V</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Collagen I coating solution</td>
		</tr>
		<tr>
			<td>Acetic Acid 0.02N, pH 3,23</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Tail Collagen (coating)</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Dispase solution</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase Stock Solution</td>
			<td>400 U/mL</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Stock solution collagenase I</td>
			<td>600 U/mL</td>
		</tr>
		<tr>
			<td>Haemolytic Solution</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>NH4Cl Solution</td>
			<td>1 parts</td>
		</tr>
		<tr>
			<td>TrisBase 20.6 g/L</td>
			<td>9 parts</td>
		</tr>
		<tr>
			<td>adjust pH to 7.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS/PS</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>Hyaluronidase Stock Solution</td>
			<td>Filter 0.2 &#181;m, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Hyaluronidase from bovine testes (powder)</td>
			<td>2,000 U/mL</td>
		</tr>
		<tr>
			<td>Sodium phosphate Buffer 1M pH7.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl Solution</td>
			<td>Store at T. amb.</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>7.1 g/L</td>
		</tr>
		<tr>
			<td>adjust pH to 7.65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorting Solution</td>
			<td>Filter 0.2 &#181;m, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.1%</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES pH 7</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Medium #1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Wash Medium #2</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Mammary 2D culture medium</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>heparin</td>
			<td>4 mg/mL</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>murine bFGF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>murine EGF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="2">Induction and Passaging of yMaSCs</td>
		</tr>
		<tr>
			<td colspan="2">Mammary Colony Medium</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>heparin</td>
			<td>4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Matrigel (add immediately before seeding)</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>murine bFGF</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>murine EGF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="2">Mammary Organoid Medium</td>
		</tr>
		<tr>
			<td>Advanced DMEM:F12</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>heparin&#160;</td>
			<td>4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>human Noggin</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>murine bEGF</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>murine EGF</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>R-Spondin 1</td>
			<td>1 &#181;g/mL</td>
		</tr>
	</tbody>
</table>
Table 1: Generation of yMaSCs. Composition of all different culture media and solutions required for isolation of primary mammary LD cells and induction of yMaSCs (section 1.)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Isolation of primary pancreatic acini</td>
		</tr>
		<tr>
			<td colspan="2">Acinar Culture Medium</td>
		</tr>
		<tr>
			<td>BPE</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.1%</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>0.1%</td>
		</tr>
		<tr>
			<td>ITS-X</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>SBTI</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>Waymouth&#8217;s Medium</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Acinar Wash Medium</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.1%</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>RPMI Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>SBTI</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td colspan="2">Acinar Recovery Medium</td>
		</tr>
		<tr>
			<td>Acinar Culture Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>30%</td>
		</tr>
		<tr>
			<td colspan="2">Collagenase I solution A</td>
		</tr>
		<tr>
			<td>Acinar Wash Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>Stock solution collagenase I</td>
			<td>360 U/mL</td>
		</tr>
		<tr>
			<td>PBS/PS</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Stock solution collagenase I</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase, type I (powder)</td>
			<td>6,000 U/mL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Passaging of pancreatic organoids</td>
		</tr>
		<tr>
			<td colspan="2">Collagenase I solution B</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td></td>
		</tr>
		<tr>
			<td>Stock solution collagenase I</td>
			<td>240 U/mL</td>
		</tr>
		<tr>
			<td colspan="2">Pancreatic Organoid Medium</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>gastrin</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>human FGF10</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>human Noggin</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>murine EGF</td>
			<td>50 ng/ml</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>1.25 mM</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>R-Spondin 1</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td>SBTI</td>
			<td>0.2 mg/mL</td>
		</tr>
	</tbody>
</table>
Table 2: Generation of yDucts. Composition of all different culture media and solutions required for isolation of primary acinar cells and induction and passaging of yDucts (section 2.)

Watch this Scientific Journal Video about De Novo Generation of Somatic Stem Cells by YAP/TAZ at JoVE.com

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient ...

de-novo-generation-of-somatic-stem-cells-by-yaptaz

Nanyang Technological University

Research

JoVE Journal

Biology

9.0K Views.  University of Padua School of Medicine. Beschikbaarheid van somatische SCs is van cruciaal belang voor regeneratieve geneeskunde, ziekte modellering en inzicht te krijgen in de eigenschappen van de SC. Hier presenteren we experimentele strategieën om te herprogrammeren, in vitro, gedifferentieerde volwassen cellen in hun overeenkomstige uitbreidbaar weefsel-specifieke stam/voorlopercellen door de voorbijgaande uitdrukking van de enkele transcriptionele co activator YAP.

Video: De Novo Generation of Somatic Stem Cells by YAP/TAZ

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Developmental Biology

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

Genetics

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent epiblast cells by orchestrated exposures to cytokines, including bone morphogenetic protein 4 (Bmp4). In human embryos, the earliest PGCs have been identified on the endodermal wall of yolk sac around the end of the 3rd week of gestation, but little is known about the process of human PGC specification and their early development. To circumvent the technical and ethical barriers of studying human embryonic PGCs, surrogate cell culture models have been recently generated from pluripotent stem cells. Here, we describe a 13-day protocol for robust production of human PGC-Like Cells (hPGCLCs). Human induced pluripotent stem cells (hiPSCs) maintained in the primed pluripotency state are incubated in the 4i na&#239;ve reprogramming medium for 48 hours, dissociated to single cells, and packed into microwells. Prolonged maintenance of hiPSCs in the na&#239;ve pluripotency state causes significant chromosomal aberrations and should be avoided. hiPSCs in the microwells are maintained for an additional 24 hours in the 4i medium to form embryoid bodies (EBs), which are then cultured in low-adherence plasticware under a rocking condition in the hPGCLC induction medium containing a high concentration of recombinant human BMP4. EBs are further cultured for up to 8 days in the rocking, non-adherent condition to obtain maximum yields of hPGCLCs. By immunohistochemistry, hPGCLCs are readily detected as cells strongly expressing OCT4 in almost all EBs exclusively on their surface. When EBs are enzymatically dissociated and subjected to FACS enrichment, hPGCLCs can be collected as CD38+ cells with up to 40-45% yield.

Primordial germ cells (PGCs) are common precursors of both sperm and eggs. Human embryonic PGCs are specified from pluripotent epiblast cells through interactions of cytokines. Here, we describe a 13-day protocol of inducing human cells transcriptomally resembling PGCs at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells.

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

Author Spotlight: Advancements in Cell and Tissue Engineering for Tendon Repair 

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...

Bioengineering

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

De Novo Generation of Somatic Stem Cells by YAP/TAZ

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