Name: de novo generation of somatic stem cells by yap/taz
Uploaded: 2018-05-07T14:00:00
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).

     

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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 border="0" cellpadding="0" cellspacing="0" width="1099">
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		<tr height="21">
			<td height="21" style="height:21px;width:481px;">10 mL sterile syringes</td>
			<td style="width:123px;">Rays</td>
			<td style="width:112px;">10LC</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm&#160; cell strainer</td>
			<td>Corning</td>
			<td>352360</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">15 mL sterile conical tubes</td>
			<td style="width:123px;">Corning</td>
			<td style="width:112px;">430052</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">24-well ultra low attachment plates</td>
			<td style="width:123px;">Costar</td>
			<td style="width:112px;">3473</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">40 mm cell strainers</td>
			<td>Corning</td>
			<td style="width:112px;">352340</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">48-well multiwell plates</td>
			<td style="width:123px;">Corning</td>
			<td style="width:112px;">353078</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">50 mL sterile conical tubes</td>
			<td style="width:123px;">Corning</td>
			<td style="width:112px;">430290</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">6-well multiwell plates</td>
			<td style="width:123px;">Corning</td>
			<td style="width:112px;">353046</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Advanced DMEM/F12</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">12634028</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 supplement (50x)</td>
			<td>Gibco</td>
			<td>17504001</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BPE</td>
			<td style="width:123px;">Gibco</td>
			<td>13028014</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Collagenase, type I</td>
			<td>Sigma</td>
			<td>17018029</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dexamethasone</td>
			<td>Sigma</td>
			<td>D4902&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">1705-041</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Disposable scalpels</td>
			<td style="width:123px;">Swann-Morton</td>
			<td style="width:112px;">0503</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">11320033</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">DMSO</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:112px;">D2650</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DnaseI</td>
			<td style="width:123px;">Roche</td>
			<td style="width:112px;">11284932001</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">doxycycline hyclate</td>
			<td>Sigma</td>
			<td>D9891&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:112px;">E5134</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 100%</td>
			<td>Sigma</td>
			<td>51976</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">FACS tubes (with strainer caps)</td>
			<td style="width:123px;">Falcon</td>
			<td style="width:112px;">352235</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td style="width:123px;">Gibco</td>
			<td>10270106</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD326 (Ep-CAM)</td>
			<td style="width:123px;">BioLegend</td>
			<td style="width:112px;">118208</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">FUdeltaGW-rtTA&#160;</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:112px;">&#160;#19780&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">FUW-tetO-EGFP</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:112px;">&#160;#84041</td>
			<td style="width:384px;">used as negative control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">FUW-tetO-MCS</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:112px;">&#160;#84008</td>
			<td style="width:384px;">used as negative control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">FUW-tetO-wtYAP</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:112px;">&#160;#84009</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:481px;">FUW-tetO-YAPS94A</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:112px;">&#160;#84010</td>
			<td style="width:384px;">used as negative control (transcriptionally dead YAP mutant)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMax</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">35050061</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">24020117</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma</td>
			<td>30721</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heparin sodium salt</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:112px;">H3149&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer solution (1M)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">15630-056</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human R-Spondin1 (His Tag)</td>
			<td>Sino Biological</td>
			<td>11083-H08H-5</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase from bovine testes</td>
			<td>Sigma</td>
			<td>H3506&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ITS-X</td>
			<td style="width:123px;">Gibco</td>
			<td>51500056</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">K-14 antibody</td>
			<td style="width:123px;">Life Technologies</td>
			<td>Ab7800&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">K-8 antibody</td>
			<td style="width:123px;">Life Technologies</td>
			<td>Ab14053&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:112px;">&#160;25030081</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lin (allophycocyanin [APC] mouse lineage antibody cocktail)</td>
			<td style="width:123px;">BD Biosciences</td>
			<td style="width:112px;">51-9003632</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel&#174; Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free</td>
			<td>Corning</td>
			<td>356231</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>J.T.Baker</td>
			<td>0402</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NH4Cl</td>
			<td>Sigma</td>
			<td>A9434&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td>Sigma</td>
			<td>72340</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">non-cell adhesive 10 cm dishes (sterile polystirol petri dish &#248; 94)</td>
			<td style="width:123px;">ROLL</td>
			<td style="width:112px;">18248</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 10x</td>
			<td>Euroclone</td>
			<td>ECM4004XL</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Hamster Anti-Mouse CD61</td>
			<td style="width:123px;">BD Biosciences</td>
			<td style="width:112px;">553347</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-Cy5 Rat Anti-Human CD49f</td>
			<td style="width:123px;">BD Biosciences</td>
			<td style="width:112px;">551129</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse/rat CD29 Antibody</td>
			<td style="width:123px;">BioLegend</td>
			<td style="width:112px;">102222</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep (10,000 U/mL)</td>
			<td style="width:123px;">Gibco</td>
			<td>15140122</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Tail Collagen I (coating)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:112px;">122-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Tail Collagen I for 3D culture</td>
			<td>Cultrex</td>
			<td>3447-020-01&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">recombinant human FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">recombinant human Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">recombinant murine EGF</td>
			<td>Peprotech</td>
			<td>315-09</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">recombinant murine FGF basic (bFGF)</td>
			<td>Peprotech</td>
			<td style="width:112px;">450-33</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium</td>
			<td style="width:123px;">Gibco</td>
			<td>31870025</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SBTI (Trypsin inhibitor from Glycine max)</td>
			<td>Sigma</td>
			<td>T6522&#160;</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris BASE&#160;</td>
			<td>Roche</td>
			<td>11814273001</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA 0,05%</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waymouth medium</td>
			<td style="width:123px;">Gibco</td>
			<td>31220023</td>
			<td style="width:384px;"></td>
		</tr>
	</tbody>
</table>

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 ...

The overall goal of this procedure is to turn primary differentiated cells into progenitor cells of the same lineage by transient expression of the transcription factor YAP. This reprogramming procedure offers the possibility to generate in vitro progenitor like or stem like cells of various tissues starting from their differentiated counterparts even when the endogenous stem cells are rare or difficult to identify. The possibility here outlying to expand somatic cells ex vivo has implications for regenerative medicine for understanding mechanisms of cell plasticity that is also relevant in cancer and for cell tissue and development of biology studies.This procedure fills a black box in the current reprogramming arena as we're not aiming to make specialized cells through embryonic stem cells differentiation, but tissue specific stem cells from differentiated cells. After sacrificing 10 female mice according to the text protocol, dissect the mammary glands by making a y shaped incision along the abdominal skin. Then use dumont forceps to carefully separate the glands from the peritoneum by gently pulling.Place the dissected glands in a non cell adhesive dish with 10 milliliters of ice cold HBSS+PS. Under a tissue culture hood use 10 milliliters of fresh HBSS+PS to wash each gland once and place them in an empty non cell adhesive dish. Then use two sterile scalpels to finely mince the glands into one cubic millimeter fragments.Proper mincing of the glands is essential for efficient cell isolation. Pull 20 glands together and try to perform this step within three to five minutes for optimal cell viability. Using a 25 milliliter serological pipette and 10 milliliters of dissociation medium, recover the minced tissue from each dish and transfer the suspension in a 50 milliliter conical tube pipetting at least five times to disaggregate the tissue clumps.Incubate the tissue at 37 degrees celsius for one hour with continuous vigorous shaking. Then spin down the digested tissue at 400 times g at room temperature for five minutes. After discarding the supernatant, use three milliliters of hemolytic solution to resuspend the tissue and incubate on ice for three minutes.Next, with 10 milliliters of wash medium number one, wash the cells. After spinning the digested tissue and discarding the supernatant, resuspend the tissue pellet in 10 milliliters of wash medium number two and plate the cells in 10 centimeter tissue culture dishes. Then incubate the dishes at 37 degrees celsius for one hour.Recover the cell suspension into a 50 milliliter conical tube and spin the tube at 400 times g for five minutes. Then use 10 milliliters of the calcium chelating solution to resuspend the pellet. Following centrifugation and after repeating the wash, resuspend the pellet in five milliliters of 0.25%trypsin-EDTA.After incubating the sample, add five milliliters of dispase solution supplemented with one microgram per milliliter of DNase one on top of the trypsin solution. To disaggregate the clumps use a one milliliter pipette tip to pipette up and down at least five times and incubate the sample at 37 degrees celsius for 10 minutes shaking every three minutes. Add 10 milliliters of wash medium number two and filter the solution through a 40 micrometer cell strainer into a new 50 milliliter conical tube.Then spin down the cell suspension. To carry out cell purification by FACS, use 200 microliters of wash medium number one to resuspend each pellet. Then add 44.5 microliters of antibody mix, pipette thoroughly, and incubate the sample in the dark on ice for 30 minutes.Dillute the cell suspension in 10 milliliters of wash medium number one and spin down the sample. Resuspend the cell pellet in two milliliters of sorting solution and filter through a cap strainer FACS tube before proceeding to FACS separation of the cell populations. Use wash solution number one to wash the cells recovered from FACS.After removing the supernatant, use mammary 2D culture medium to resuspend the cell pellet and plate 500 microliters per well on a collagen treated plate. Seven days after doxycycline induction of lentiviral infected primary LD cells, add 150 microliters per well of 05%trypsin-EDTA to the cells and incubate them at 37 degrees celsius for 10 minutes. Once the cells have been washed and counted, use memory colony medium, supplemented with two micrograms per milliliter of doxycycline to resuspend them.Then seed 1, 000 cells per well in 24 well ultra low attachment plates for culturing YAP expressing mask like colonies. To subculture y masks, collect each sample and add a 10 to one volume of ice cold HBSS, then incubate the samples on ice for one hour in order to solubilize the basement membrane matrix. After spinning down the colonies, use ice cold HBSS to wash the cells three times.Then add 05%trypsin-EDTA to the colonies and incubate them at 37 degrees celsius for 10 minutes. Pass the colonies through a P1000 pipette tip 10 times to obtain a single cell suspension. Then count and reseed the cells in mammary colony medium without doxycycline in 24 well ultra low attachment plates at a colonogenic density of 1, 000 cells per well.Prior to the third passage, transfer y mask colonies in organoid culture conditions by recovering the colonies from the mammary colony medium and use 100%growth factor reduced basement membrane matrix to resuspend them. Following incubation when the basement membrane matrix has solidified overlay the gels with 500 microliters of the mammary organoid medium. Passage the cultures by collecting each organoid sample and incubating them in an excess volume of HBSS on ice for one hour.Then use ice cold HBSS to wash the organoids three times. Trypsinize the organoids as demonstrated earlier in this video and reseed single cell suspensions in a drop of 100%growth factor reduced basement membrane matrix, then use 500 microliters of mammary organoid medium to overlay the gels. After washing digested mouse pancreatic tissue according to the text protocol, use six milliliters of acinar recovery medium to carefully resuspend the digested tissue and split it between two wells of a six well tissue culture plate.Under a stereomicroscope carefully assess the quality of the acinar isolation and remove any big tissue clumps eventually present. To seed primary pancreatic acini, after incubating the clusters for two hours for cell recovery, collect the acinar cell suspension in a conical tube and centrifuge the samples at 100 times g and 18 degrees celsius for five minutes. After resuspending acini in acinar culture medium dilute the suspension with an equal volume of neutralized rat tail collagen one solution, keeping the tubes on ice.Mix carefully and quickly and seed the cell suspension in 16 wells on top of the collagen cushion. For optimal seeding of pancreatic acini it is crucial to keep all tubes in ice before plating and proceed with speed but at the same time careful pipetting to avoid air bubble formation is essential to create a homogenous collagen matrix. Incubate the cells at 37 degrees celsius for one hour to allow a hydrogel to form.Then to induce pancreatic organoids, overlay the collagen hydrogels with 500 microliters of acinar culture medium supplemented with doxycycline. To subculture the organoids, discard the culture medium, carefully extract the hydrogels, and transfer them to conical tubes containing four milliliters of collagenase I solution B then incubate the tubes at 37 degrees celsius with vigorous shaking for 30 minutes before spinning down the cells and removing the supernatant. After trypsinizing the cells and removing the PBS supernatant, use ice cold basement membrane matrix to resuspend the cell pellet and seed the suspension in ultra low attachment plates.Let the basement membrane matrix hydrogel solidify by incubating the plates at 37 degrees celsius for 40 minutes and then overlay with pancreatic organoid medium. yDucts will grow as cyst like organoids in seven to 10 days. Careful gating of primary mammary LD epithelial cells into the three subpopulations shown here 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.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. Under basement membrane matrix organoid culture conditions, reprogrammed luminal cells self organize into complex organoid like structures that develop around multiple lumens and self renew even in the absence of doxycycline. Within the five to seven days of culture in 3D collagen I based hydrogel in the presence of doxycycline, differentiated panrcreatic acini readily turn into duct like clusters or yDucts composed of a thin monolayer of epithelial cells that proliferate around an expanding central cavity.The reprogramming efficiency can be easily measured by scoring the number of duct like clusters over the total number of seeded acini. Reprogrammed yDucts can then be passaged at the single cell level into matrigel based organoid culture conditions displaying remarkable self renewal ability even in the absence of endogenous YAP expression. We have detailed two procedures.One allowed reprogramming of FACS purified cells through lentiviral vectors and a second one that avoids viral infection and takes advantage of transgenic YAP expression. While attempting reprogramming of mammary cells it is important to avoid the use of excessive viral titer as this can be detrimental for reprogramming efficiency. The fully transgenic strategy is appropriate for primary pancreatic acini as these are scarcely amenable to lentiviral infection.It also offers the same advantage of doxycycline dependent lentiviral vectors for the type control of gene expression. In addition to the exampes demonstrated here, this YAP reprogramming protocol has proven successful for converting differentiated neurons into proliferating neurons themselves. After watching this video you should have a good understanding on how to perform a YAP dependent reprogramming to convert differentiated cell types from different adult tissues into their corresponding tissue specific stem cells.This procedure expands the current reprogramming strategies by providing a means to generate somatic stem cells that may have relevance for regenerative medicine for the study of somatic stemness and for expansion of somatic stem cells in vitro. Please consider that handling of lentiviral preparation can be extremely hazardous and by safety level two measures should be always adopted while performing this procedure.

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