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">
	<tbody>
		<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 ...

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8.9K Views.  University of Padua School of Medicine. 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.

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

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.