The fact that hub regions

The fact that hub regions constitute potential points of vulnerability for network disintegration is a key issue (Albert et al., 2000). Indeed, the abnormalities associated with several SAG Supplier disorders have been shown to concentrate in hubs; such disorders include ADHD (dos Santos Siqueira et al., 2014), autism (Ray et al., 2014) and schizophrenia (Tomasi and Volkow, 2014). As such, hub disruption has been proposed as a general pathological mechanism in brain disorders (Crossley et al., 2014; van den Heuvel and Sporns, 2013). Therefore, description of atypical neurodevelopmental trajectories of the brain connectome appears crucial to unveil the neural substrates of mental symptoms and disorders (Di Martino et al., 2014). However, full characterization of these functional networks and possible differences in their architecture in childhood has not yet been achieved.

Materials and methods

Fig. 1 highlights a strong replication (Pearson’s r=0.91, p<0.001) of the ranks of the mean EVC (across subjects) for brain regions in the two sites, especially for regions with high and low EVC values; more variability was observed in the intermediate values. Our main result was the overlap (across sites) of 21 regions within the set of regions with EVC scores within the top 10% (see Table 2). For the top 5% and 1%, the number of overlapping regions was 11 and 2, respectively. The p-value for the comparison of the number of overlapping regions was less than 0.001 (Monte Carlo method), regardless of the threshold (10%, 5% and 1%) considered. As shown in Fig. 2, the individual variability in the EVC scores of the overlapping regions was relatively low, with similar results for both sites. Fig. 3 shows brain maps of the hub regions that overlapped across the sites. Note that regardless of the threshold considered, the anterior medial prefrontal cortex (amPFC) and the IPL/intraparietal sulcus (IPS) were included among the most relevant regions. Regarding the GLM analyses of psychopathological manifestations, only four regions were not strongly affected by the inclusion of head motion (FD) as a covariate (Table 3). Among these four regions, the EVC rank of the right intraparietal cortex (see Fig. 4) was found to be negatively associated with the CBCL score, a finding replicated at both sites (uncorrected p<0.02 and 0.03 for site 1 and site 2, respectively). The box plots presented in Fig. 4 (bottom) were built considering a quartile categorization of the mean EVC rank (low if less than first quartile; high if greater than third quartile; typical otherwise). The data were categorized this way solely for visualization purposes (the statistical evaluation was carried out previously through a GLM in which EVC was considered as a quantitative variable) because the association effect is more evident in box plots than in scatter plots. Regarding head motion, the mean FD (± sd) was 0.19 (±0.29) for site 1 and 0.12 (±0.12) for site 2. As expected, the EVC of the top 10% of regions was significantly correlated (Pearson correlation) with the mean FD. However, the Pearson correlation between mean FD and CBCL score was not significant (p=0.40 and 0.21 for site 1 and 2, respectively). In addition, we repeated both the hub identification and CBCL analyses by considering only the subjects with a mean FD<0.2 mm. Only one of the previous 21 regions was not identified as a replicable hub (the right Insula). The results of the CBCL analysis remained the same (see Supplementary Table S1 for further details). We did not find any significant association between EVC and estimated IQ in either of the two sites. Finally, the findings were not altered by the inclusion of socioeconomic status (SES) as an additional nuisance variable in the GLM analyses (Supplementary Table S2).
In addition to the IPL/IPS and amPFC, other fairly consistent regions (across gender and age) included the ventral medial prefrontal cortex (vmPFC), medial temporal gyrus (mTG), precuneus and PCC. Many of these regions are components of the default mode network (DMN) (Raichle et al., 2001; Raichle and Snyder, 2007). The IPL/IPS and amPFC are associative areas and might play a role as hubs in the cross-modal integration of cortical and subcortical regions (Buckner et al., 2008). The amPFC is involved in social cognition (Wang and Hamilton, 2014), metacognitive abilities for memory and perception (Baird et al., 2013), evaluative judgment and self-referential processes (Zysset et al., 2003). The IPS/IPL is part of the parietal cortex (Rademacher et al., 1992), which has been characterized as a cross-modal hub of multisensory information convergence (Seghier, 2013) and implicated in several cognitive processes. Semantic processing and complex language functions frequently activate the angular gyrus, for instance (Binder et al., 2009; Vigneau et al., 2006). In addition, the right IPL is also involved in attentional maintenance, the encoding of salient events (Singh-Curry and Husain, 2009), spatial cognition, internal representations (Sack, 2009), social cognition (Buckner et al., 2008) and the autobiographical memory system (Spreng et al., 2009). Thus, our findings support crucial roles for the IPL/IPS and amPFC as integrative regions.

The compositions of different energetic metallic particles and corresponding

The compositions of different energetic metallic particles and corresponding coatings are chosen in order to take advantage of the resulting JQ1 Supplier reactions of alloying when the metals are combined or alloyed through heat activation. Bimetallic particles composed of a core/shell type structure of having different metals are to be properly chosen so that upon achieving the melting point (for at least one of the metals) a relatively great deal of exothermic heat of alloying is liberated. In a typical embodiment, the core metal is aluminum and the shell metal is nickel. Throughout the coating process the nickel may be deposited onto the outer surface of the aluminum particles by using an electrolysis process of a suitable metal salt solution with a reducing agent in an aqueous solution or a solvent media. The aluminum particles may be pretreated with zinc to remove any aluminum oxide present on the surface. The resulting bimetallic particles may be utilized as an enhanced blast additive by being dispersed within an explosive material [33]. The core metal can be one of aluminum, magnesium, boron, silicon, hafnium, or carbon. The outer shell metal is from nickel, zirconium, boron, titanium, sulfur, selenium, or vanadium. In the first stage of the procedure, 11 mL of zincate solution is mixed (a zinc gluconate solution having an approximately pH of 13) with 100 mL of deionized water. In the next step, the solution is stirred rapidly (with a magnetic PTFE stirbar) and the solution is brought to 65 °C. Then 0.25 g of aluminum powder composite is added (specifically, the grade H-60 aluminum powder). Then, the solution is stirred for 45 s, and vacuum filtered through a 1.2 µm PTFE membrane. Finally, the collected zinc coated aluminum particles are rinsed with deionized water. In the second stage, those pretreated aluminum particles are nickel plated. For this step, 30 mL of nickel sulfate is mixed with 90 mL of solution B (sodium hypophosphite), stirred with a PTFE coated stirbar and then heated to approximately 90–95 °C. Next, 0.29 g of the zinc treated aluminum powder is added and this temperature is maintained and the mixture is stirred until the appropriate amount of nickel is deposited. Then the solution is vacuum filtered through a 1.2 µm PTFE membrane. Finally, the collected aluminum core/nickel shell particles are rinsed with water, and then allowed to dry. The explosive material may be any type of explosive material that can mix with the bimetallic particles of the present invention as an enhanced-blast additive, e.g., octogen (HMX), hexahydrotrinitrotriazine (RDX), pentaerythritol tetranitrate (PETN), picrate salts and esters, dinitrobenzofuroxen and its salts, hexanitrohexaazaisowurtzitane (C-20), trinitrotoluene (TNT), glycidyl azide polymer (GAP), diazodinitrophenol (DDNP), lead azide and other azide salts, lead styphnate and other styphnate salts, triaminoguanidine nitrate, tetranitrodibenzole trazapentalente, diaminohexanitrophenyl, triaminotrinitrotoluene (TATB), or plastic bonded explosives (PBX) [33].
A processing technique was demonstrated by Vasylkiv et al., which was based on the synthesis of ceramic nanopowders and simultaneous impregnation with metallic nanoparticles by multiple “nano-blasts” of embedded cyclotrimethylene trinitramine (RDX) in preliminary engineered multi-component nano-reactors [62]. The “nano-blasts” of impregnated RDX deagglomerate the nanopowder due to the high energetic impacts of the blast waves, while in the decomposition of compounds, their solid-solubility is enhanced by the extremely high local temperature generated during the nano-explosions. The investigators applied this technique to produce nanosized agglomerate-free 8 mol% yttria-doped cubic zirconia aggregates with an average size of 53 nm impregnated with 10 mass% of platinum particles of 2–14 nm.
The same authors also published a similar article to demonstrate a unique processing technique which was based on engineering of the multi-component ceramic nanopowders and composites with precise morphology by nano-explosive deagglomeration/calcinations [63]. As mentioned above, multiple nanoexplosions of impregnated cyclotrimethylene trinitramine (RDX) deagglomerate the nanopowder (due to the highly energetic impacts of the blast waves) while the solid-solubility of one component into the other is enhanced by the extremely high local temperature generated during the nano-explosions. They applied this technique to produce nanosize agglomerate-free ceriagadolinia solid solution powder with uniform morphology and an average aggregate size of 32 nm, and as mentioned before, 8 mol% yttria-doped zirconia aggregates with an average size of 53 nm impregnated with platinum (2–14 nm).

Esta conjunci n entre un orden social

Esta conjunción entre un orden social arcaico y el capo de las drogas y su territorio se recrea en de Yuri , una de las contribuciones más interesantes MK-571 sodium salt hydrate la creciente literatura del norte. Lo que suele entenderse como narcoliteratura suele tender a adquirir la forma de historias de aventuras al estilo de de Arturo Pérez Reverte; no obstante, la novela de Herrera recrea el narcogobierno como un retroceso a una era similar a la Edad Media europea. Su protagonista es el juglar, el romancero, reclutado por el Rey y renombrado como el Artista. Éste vive en la corte junto a las cortesanas, los miembros del cartel, los sirvientes, el periodista, la Bruja y su hija, la Cualquiera y el Heredero. El término no parece descabellado cuando examinamos la compleja jerarquía y el poder sobre la vida que se le atribuye al gran barón. En el episodio que abre la novela, el cual tiene lugar en un bar, el rey ejecuta sumariamente a un borracho molesto, con lo cual despliega su poder sobre la vida ajena. Como trovador, Lobo, ahora conocido como “el Artista”, compone canciones de alabanza y baladas para conmemorar las hazañas del monarca. Estas son narcocorridos, un género musical que, al igual que otros corridos, relata una historia; uno de estos impecables resúmenes, “Contrabando y traición”, en las primeras estrofas retrata a los traficantes como héroes trágicos, al igual que “La banda del carro colorado” de Paulino Vargas:
Y así, en tres versos, nos cuenta la historia entera. El corrido es un género que refuerza la conexión que Segato destaca entre lo arcaico (la tradición de las baladas) y lo posmoderno. Al principio de la novela, Herrera vincula explícitamente lo moderno (el cine) con la transmisión de formas arcaicas de poder: “La única vez que Lobo fue al cine vio una película donde aparecía otro hombre así: fuerte, suntuoso, con poder sobre las cosas del mundo. Era un rey”. El narcorreino está situado apenas al sur de la frontera, rodeado por el desierto, “reventaba un confín del desierto en una soberbia de murallas, rejas y jardines vastísimos”, cual fortaleza medieval. Sus habitantes no son designados por patronímicos, sino por ocupación, edad u otros atributos, práctica que Herrera adopta también en otras novelas. Por lo tanto, los súbditos del reino son el Artista, el Joyero, la Niña, la Cualquiera, el Periodista y, por supuesto, el Traidor. Las justas son con armas de fuego, no con lanzas. Los comunes se acercan al señor durante las para pedirle favores personales, y el palacio está rodeado por una valla electrificada para que el reino quede separado de las comunidades circundantes, todo lo cual subraya la insistencia de Herrera en la estructura feudal.

En las crónicas y ensayos que Carlos Monsiváis escribió sobre la disidencia sexual, el ambiente gay y la historia de la homosexualidad en México se delinea lo que quisiera llamar una epistemología de un coleccionista. Dada la ausencia de un sólido de investigaciones y de datos sobre estos temas, Monsiváis reúne y analiza fragmentos de una historia subalterna y subterránea. Indaga —un coleccionista es una especie híbrida entre el archivista y el bibliógrafo— en los restos de una memoria colectiva apenas sugerida; explora vidas infames, como las llamara , y relatos menores. Y con ellos, arma pacientemente una crónica detallada y aguda de la homosexualidad en el país durante el siglo .
Si su epistemología es la de un coleccionista, su tono es el de un traductor que lee estos trozos y logra articularlos con una serie de discusiones contemporáneas sobre sexualidad, moralidad, estética, literatura y política. Monsiváis, fiel a flora esta epistemología, lee los intersticios de una cultura (la letrada y la popular) para convertirse en un pedagogo de las identidades colectivas. Y en este sentido usa la crónica como una escritura que no necesita relaciones causales ni pruebas empíricas, sino propuestas de lecturas. La suya es una historia oblicua que cruza los aparatos de citas, las fuentes y los autores canónicos para trazar la única línea posible que permitirá construir o relatar algo así como la historia de la homosexualidad en México: una diagonal que se sostiene en un vacío persistente y que encuentra en el retruécano, y en una intertextualidad por descifrar, una estrategia de escritura y de pensamiento. Gnosis minoritaria, hermenéutica de los expulsados, recibidos o rechazados. Cualquier biografía se puede convertir en una genealogía de procesos colectivos de identificación y clasificación, de formas sociales de exclusión y oclusión. Las partes que atamos a un relato que entra en la pretendida coherencia de las narrativas personales guardan el fragor de las luchas colectivas que suman y dividen, que parten y vuelven a juntar: emociones con identidades, destinos con biografías, cuerpos con deseos, descripciones con juicios, escondites con intemperies, formas con contenidos. Creo que de este modo Monsiváis convierte la escritura, al menos en este campo, en una práctica de sí que le permite hablar consigo mismo sin decir nada personal, describir a los otros citándose a sí mismo, estar presente en el sesgo y en la cita. Pero una práctica de sí no es una biografía, sino una forma de actuar en el campo de las subjetividades posibles, de crear nuevas subjetivaciones y otros enunciados, además de los que ya conocemos y por los que nos piden cuentas.

The elbow joint was immobilized

The elbow joint was immobilized using a splint postoperatively. Intravenous prostaglandin E1 was administered for 3 days to prevent thrombosis recurrence. Moreover, ultrasonography conducted on Postoperative Day 5 showed a patent flow in the left brachial, proximal radial, and ulnar arteries, without residual thrombi. No postoperative complication was observed. The preoperative bilateral hand skin temperatures remained nearly identical to the postoperative skin temperatures (Table 1).

Previous studies have reported that arterial occlusions, particularly occlusion of the axillary artery, in throwing athletes mainly occurs in the shoulder region. The axillary artery can be compressed by the humeral head and at the thoracic outlet when the arm is in the pitching position. Rohrer et al postulated that thrombosis of the axillary artery could be caused by a repetitive mechanical trauma of the humeral head during throwing.
Thoracic outlet syndrome (TOS) has been reported as the leading cause of arterial occlusion in throwers, and is described as a nrf2 inhibitor at the superior thoracic outlet caused by external pressure on the neurovascular bundle. Duwayri et al conducted a retrospective study and reported that 8.2% of TOS was arterial occlusion involving the subclavian artery (81%) and axillary artery (19%).
Duwayri et al examined arterial occlusion in their patients with TOS and reported that a spectrum of pathology could be found in overhead athletes. TOS is caused by a repetitive positional compression of the axillary artery during throwing. The pathology of vascular occlusion includes focal intimal hyperplasia, aneurysm formation, segmental dissection, and branch vessel aneurysms.
Other causes of arterial occlusion in the shoulder region include structural abnormalities such as a congenital first rib, trauma, coagulopathy, connective tissue disease, arrhythmia, and iatrogenic factors.
The cause of arterial occlusion in this patient remains obscure. Nuber et al examined upper extremity ischemia in athletes, and reported that soft tissue hypertrophy and hyperactivity played unique and predominant roles in the pathogenesis of an arterial injury. In our patient, one possible mechanism was temporary vascular compression of the adjacent swollen muscles and soft tissues induced by repetitive pitching. Stasis of the blood stream caused the formation of thrombi and complete occlusion of the arteries.

A small bowel intussusception is rare and can be caused by infection, polyp, lymphoma, vasculitis, Meckel\’s diverticulum, intestinal duplication, cystic fibrosis, and intramural hematoma. Multiple simultaneous small bowel intussusceptions are rarer. Rapunzel syndrome is an extremely rare intestinal condition resulting from a trichobezoar with a tail-like extension from the stomach to the small bowel. Rapunzel syndrome may present as chronic abdominal pain, malabsorption, gastrointestinal tract obstruction, gastrointestinal bleeding, and intussusceptions. Few cases of Rapunzel syndrome in patients presenting with intussusceptions have been reported. We report a case of a 5-year-old girl with Rapunzel syndrome causing intussusceptions and bowel obstruction.

Case Report
A 5-year-old girl presented with severe abdominal pain and nausea that started 2 days before presentation. She experienced cramping pain in the left abdomen for three weeks without other gastrointestinal symptoms. On physical examination, a left palpable abdominal mass was noted. Abdominal echo revealed typical imaging findings but an unusual location for intussusceptions. Abdominal computerized tomography (CT) revealed multiple target signs (Figure 1) over the jejunum. Radiological reduction was unsuitable for the multiple jejunal intussusceptions; therefore, laparoscopic exploration was performed. During operation, 8 segmental jejunal intussusceptions ranging from 2 cm to 8 cm in length were observed at a distance of 20–100 cm from the ligament of Treitz. All intussusceptions were laparoscopically reduced. A mass or diverticulum was not observed. The patient had a smooth recovery and was discharged on the 5th day after operation.

br Results br Discussion Recent advances enabling the


Recent advances enabling the prospective isolation of mouse satellite Doxorubicin HCl have facilitated mechanistic analyses of their myogenic function. For example, the ability to clonally sort satellite cells with high purity made possible the demonstration that these cells can undergo asymmetric division (Kuang et al., 2007; Rocheteau et al., 2012) and repopulate the satellite cell niche in vivo (Cerletti et al., 2008). While findings in mouse models are often extrapolated to human biology, whether mouse and human myogenic cells exhibit fully equivalent properties may still be questioned, particularly given significant phenotypic discrepancies in several mouse models of human muscle disease (Bulfield et al., 1984). All of these issues can be addressed through the establishment of robust methods for direct purification of human muscle progenitors.
Previous work by Pisani et al. demonstrated the utility of the sialomucin CD34 to enrich for myogenic cells within the CD34− subset of magnetically separated cells in adult muscle (Pisani et al., 2010b), consistent with immunohistochemical studies reporting the absence of CD34 in adult human muscle cells located in the satellite cell position (Lecourt et al., 2010). Pisani et al. also noted mixed myogenic and adipogenic activity within CD34+ adult muscle cells, which showed differential expression of CD56 (Pisani et al., 2010a). Findings from our study confirm that CD34 distinguishes myogenic and nonmyogenic cells within the nonhematopoietic, nonendothelial (CD45−CD11b−GlyA−CD31−) hMFA cell pool in both fetal and adult tissue: CD34+ cells are PAX7-negative, adipogenic cells that do not possess any myogenic activity, whereas within the CD45−CD11b−GlyA−CD31−CD34−/low subset, selection of CD56intITGA7hi cells yields a highly enriched population of PAX7-expressing, robustly myogenic progenitors. Yet, it is important to note that these FACS-based strategies pertain to cells isolated from fresh muscle only. Sorted cells may undergo marked changes in their surface marker profiles during ex vivo culture, and it is unclear if our protocols are applicable to cells that have undergone expansion/differentiation in culture.
Fluorescence-activated cell-sorted fetal human CD34−CD56intITGA7hi cells engraft in mouse muscle to form new myofibers, albeit at low efficiency (Figures 5B–5F). Low-level engraftment of human cells into mouse tissue is not unexpected, as similar outcomes have been observed for other human, tissue-specific stem and progenitor cells upon transfer into immune-compromised mice (Doulatov et al., 2012; Racki et al., 2010). Unfortunately, given the relatively sparse presence of human myofibers in this system, we were unable to establish conditions to reliably detect costaining for PAX7 and human species-specific nuclear antigens on engrafted mouse muscle sections. We therefore were unable to determine if fluorescence-activated cell sorted fetal human CD34−CD56intITGA7hi cells can repopulate the PAX7-expressing satellite cell pool in vivo. We also were unable to test the in vivo engraftment potential of fluorescence-activated cell-sorted progenitors from adult human muscle, given the low yield of cells that could be obtained from adults (Figure 7E).
Interestingly, enrichment of PAX7 expression and myogenic activity within the CD34− compartment in human muscle stands in contrast to immunophenotyping studies in mouse muscle, which localize myogenic activity to the CD34+ subset of mouse MFA cells (Beauchamp et al., 2000; Conboy et al., 2010; Montarras et al., 2005; Sacco et al., 2008; Sherwood et al., 2004). Species-specific differences in CD34 expression have also been noted in other somatic stem cell populations, including hematopoietic stem cells (HSCs), which are CD34+ in adult human bone marrow and CD34− in adult mouse bone marrow (Okuno et al., 2002; Osawa et al., 1996). Such differences appear to arise from the presence of species-specific upstream regulatory elements, which differentially regulate CD34 gene transcription in mouse and human cells (Okuno et al., 2002).

To test the efficiency of gene knockdown

To test the efficiency of gene knockdown, the ES cell lines expressing the shPU.1 constructs were differentiated into hematopoietic cells. The transcription factor PU.1 is not expressed in ES e1 activating enzyme at day of differentiation and becomes expressed by day 9 when hematopoietic progenitor cells are generated (Fig. 3A). At this time point, the expression of PU.1 in the two knockdown cell lines was >80% lower than expression in the control GFP-expressing ES cell line. The day 9 hematopoietic progenitor cells were then put in liquid culture for an additional four days in a cytokine cocktail favoring myeloid cell generation. At day 13 of differentiation, the expression of PU.1 in the two knockdown lines was >90% lower than expression in the control cell line (Fig. 3A). These data demonstrate that constitutively expressed AAVS1-targeted knockdown constructs against PU.1 can successfully decrease expression levels throughout differentiation.
To determine the effect of decreased PU.1 levels on hematopoietic cell development, day 9 progenitor cells were put into liquid expansion cultures and analyzed for erythroid (CD235+CD41−) and myeloid (CD18+CD45+) lineages. Both knockdown cell lines expressed almost undetectable levels of CD18 and CD45 compared to ~50% co-expression of these markers on control GFP-expressing cells, indicative of a block in myelopoiesis (Fig. 3B). Furthermore, the absolute yield of myeloid cells in the two knockdown lines was much lower compared to the GFP control cell line (Fig. 3C). These findings were confirmed by gene expression analysis of the myeloid marker cFMS, which was also drastically reduced in the PU.1 knockdown cell lines (Fig. 3D). Analysis of the erythroid lineage demonstrated an ~4-fold expansion in the percentage of CD235+ cells (Fig. 3B) with a concomitant increase in absolute yield of cells, especially in the knockdown line set 2 (Fig. 3C). These findings were confirmed by an increase in gene expression of the erythrocyte transcription factor EKLF in the two knockdown cell lines (Fig. 3D). The pan-hematopoietic marker GATA1 was unaffected and there was a minor increase in the megakaryocyte marker PF4. These data demonstrate that AAVS1-targeted knockdown of PU.1 specifically decreases the myeloid lineage and skews the progenitor cells to an erythroid cell fate.

The CD43 promoter fragment we describe here allows tissue specific, controlled expression of a transgene specifically in cells of the hematopoietic lineages. During the differentiation of the CD43-GFP hematopoietic progenitors into the erythroid lineage it was observed that these cells began to lose the expression of GFP over time in culture. Since endogenous CD43 also decreases, this observation is most likely due to increased sensitivity of the CD43 antibody over GFP and not due to silencing of transgene expression. It has been shown that mature erythrocytes lose expression of CD43, hence this loss of expression is anticipated (Remold-O\’Donnell et al., 1987). Importantly, myeloid cells, which maintain CD43 expression in the adult, do e1 activating enzyme not lose GFP expression in the reporter lines while in culture (Fig. 2C). The CD43 reporter represents a valuable tool to easily follow hematopoietic specification from ESCs in live cultures with specificity that rivals CD41 or CD45 as all hematopoietic progenitors are CD43+ (Vodyanik et al., 2006).
In mouse models, a homozygous loss of PU.1 is embryonic lethal (Scott et al., 1994) or if mice are born alive, they die shortly after birth (McKercher et al., 1996). Mice that are born are found to lack any mature B and T cells, macrophages and neutrophils but have erythrocytes and megakaryocytes (McKercher et al., 1996) indicating that PU.1 is required for myeloid cell lineage development. A heterozygous loss of PU.1 is non-lethal, however it leads to a loss in the formation of multipotent myeloid progenitors (Scott et al., 1994). We demonstrate that a >80% knockdown of PU.1 in human hematopoietic cells results in a loss of myeloid lineage cells, phenocopying what occurs with a loss of PU.1 in the mouse. The differentiation of ES cells into primitive hematopoietic cells can be considered to mimic human development, thus our data suggests that during early human hematopoiesis PU.1 is also required for myeloid lineage development.

br Results br Discussion We


We have established a novel serum-free culture method for generating and expanding CD271+CD73+SOX9+ chondrogenic ectomesenchymal EPZ015666 from hPSC-derived, CD271hiCD73− neural crest-like progeny. The differentiation pathways from hPSCs to chondrocytes via chondrogenic ectomesenchymal cells postulated from this study are summarized in the Graphical Abstract. Under optimal conditions, the generation of neural crest-like progeny from hPSCs in CDM was achieved more quickly than previously reported, and the resultant ectomesenchymal cells displayed long-term chondrogenic activity.
Quantitative data on the efficiency of genesis of SOX9-expressing chondroprogenitors from hESCs were first shown by Oldershaw et al. (2010). Under their conditions in which hESC differentiation is directed initially to mesoderm then toward chondrocytes, approximately 8.5 chondroprogenitors of 75%–97% SOX9+ are produced per hESC by day 14 of differentiation. In contrast, while only about 0.6 CD271hiCD73− neural crest-like progeny were generated per hPSC by day 6 of differentiation in CDM plus SB431542 (data not shown), our method for neural crest-like cell culture in CDM in the presence of FGF2+SB431542 led to 19 to 38 CD271+CD73+ chondroprogenitors per hPSC by day 14 (Figure S3A). Approximately 76%–85% of these cells continued to express SOX9 protein on days 27–33 (p8–p10), a proportion that increased slightly to about 97% after 3 days of treatment with FGF2+TGFβ3. Further expansion yielded approximately 1 × 107 chondrogenic ectomesenchymal cells per hPSC by day 51 (p16, 24 population doublings). Thus, the method described here allows human chondrogenic cells to expand for an extended period without the loss of purity and chondrogenic activity, resulting in a much larger yield of chondroprogenitor cells (although not homogenous) from hPSCs than previously attained.
The effect of SB431542 on expansion of endothelial progenitors derived from mouse and human ESCs has also been reported (James et al., 2010; Watabe et al., 2003). A lack of Nodal/Activin/TGFβ signaling may generally promote proliferation and prevent terminal differentiation of embryonic stem/progenitor cells. In this respect, the ways in which FGF2+SB431542 helps to maintain SOX9 and CD271 while suppressing N-cadherin expression during expansion of the chondrogenic CD271+CD73+ cells and in which CD271hiCD73− neural crest-like cells are directed to give rise to such cells are interesting topics for future study. In this respect, comparative transcriptome analysis has revealed that the CD271+CD73+ cells accumulated under FGF2+SB431542 conditions possess a similar mRNA profile to primitive neural crest/ectomesenchymal cells, although they lacked SOX10 expression, which is critical for neural and melanocytic lineage commitment. Thus, suppression of Nodal/Activin/TGFβ signaling does not seem to freeze the developmental stage of the hPSC-derived neural crest during expansion. Such suppression may instead simply support the high proliferative potential of the cells as well as the expression of SOX9 (and COL2A1), and thereby maintain chondrogenic activity. SOX9 expression initiated at the specification and premigratory stages is transient in trunk neural crest but persists in cranial neural crest (Cheung and Briscoe, 2003; Cheung et al., 2005; McKeown et al., 2005). The chondrogenic CD271+CD73+ ectomesenchymal cells that maintain SOX9 transcription and translation (SOX9-GFP+/lo) may therefore represent proliferating cranial neural crest, with a slight commitment to non-neural lineages.
Genesis of MSC-like cells from hESCs directly or via neuroectoderm specification has been demonstrated (Chambers et al., 2009; Mahmood et al., 2010). As shown in Figure S4D, the colonogenic (CFU-F) activity, representing self-renewal activity of MSCs, emerged as early as p2 of the expansion culture under FGF2 alone (i.e., in CD271−CD73 cells) or FGF2+SB431542 (i.e., in CD271+CD73+ cells). However, we were unable to demonstrate significant adipogenic or osteogenic activity in either cell type (data not shown), although the GO analysis predicted osteogenic activity in cells generated under FGF2 (data not shown) and FGF2+SB431542→FGF2 conditions. This discrepancy may be due to some degree of heterogeneity in the cell types generated under these conditions or to the assay method employed, which is widely used for MSCs (Pittenger et al., 1999), but is less well validated for mesenchymal cells derived from human neural crest. Furthermore, the FGF2+SB431542 conditions applied to the expansion culture of mouse bone marrow MSCs resulted in inhibition of proliferation and promotion of adipocytic differentiation (data not shown). The CD271+CD73+ ectomesenchymal cells are therefore very likely not to be MSCs, although the possibility that they are derived from an MSC-like precursor has not been excluded.

Introduction The adult intestinal epithelium

The ctap adult intestinal epithelium is a self-renewing tissue with a high turnover rate maintained by intestinal stem cells that reside at the ctap of glands (called crypts). Lgr5 (leucine-rich-repeat-containing G protein-coupled receptor 5), a Wnt/β-catenin target gene, exclusively marks these long-lived crypt-based columnar (CBC) stem cells in the mouse and human intestine (Barker, 2014; Barker et al., 2007; Itzkovitz et al., 2012). Wnt/β-catenin signaling is crucial for normal stem cell function in the intestinal epithelium (Korinek et al., 1998; Sato et al., 2009). More specifically, Wnt3 signaling, provided by flanking Paneth cells, is necessary for the maintenance and function of CBC stem cells (Sato et al., 2011). In the absence of Wnt3, Wnt2b can compensate (Farin et al., 2012). The weak short range Wnt signal is augmented by R-spondin signaling through Lgr receptors (Carmon et al., 2011; de Lau et al., 2011). R-spondins are incorporated into a complex that contains Lrp (low-density lipoprotein receptor-related proteins), Lgr, and Fzd (Frizzled); this complex facilitates Fzd-coupled Wnt/β-catenin signaling. Although studies show that Wnt is critical for stem cell function (Farin et al., 2012; Sato et al., 2011), other studies question the requirement for secreted Wnt and the source of Wnt in vivo (for example, San Roman et al., 2014). Here we circumvent these controversies by investigating Fzd function.
Of the ten mammalian Fzds, only Fzd7 is frequently upregulated in stem cell populations and cancers from diverse tissues (Vincan and Barker, 2008). Cell fractionation (Mariadason et al., 2005) and in situ mRNA expression (Gregorieff et al., 2005) studies show that Fzd7 is at the base of intestinal crypts, the correct location to transmit stem cell Wnt signals. Using tissue- and cell-specific gene deletion, we demonstrate that Wnt-dependent Lgr5+ stem cell processes are impaired in the absence of Fzd7.


Taken together, our data show that Fzd7, at least in part, transmits the critical Wnt signal in intestinal CBC stem cells. Expression of Fzd7 was enriched in Lgr5+ stem cells, and Fzd7 signaling was necessary for competent Lgr5+ stem cell function, as in the absence of Fzd7, crypt homeostasis and epithelium regeneration were compromised. Closely related Fzds could not fully compensate for Fzd7 loss.
We observed a rapid loss of CBC cells 1 day after Fzd7 deletion in induced AhCre;Fzd7 mice. By counting PCNA+ cells between the Paneth cells or unstained CBC cells between cryptidin stained Paneth cells, as surrogate markers for CBCs, we avoid the documented pitfalls of stem cell marker specificity (Barker, 2014; Itzkovitz et al., 2012; Metcalfe et al., 2014). Recombination using with Lgr5Cre mice confirmed that deletion of Fzd7 specifically in Lgr5+ cells was deleterious to these intestinal stem cells. Similar rapid loss of stem cells following deleterious gene deletions was observed previously with c-Myc (Muncan et al., 2006) and Ascl2 (van der Flier et al., 2009), for example. The epithelium recovers rapidly from these deleterious insults in vivo. Two mechanisms have been described recently to account for the rapid repopulation with stem cells: dedifferentiation of partially committed progenitors of the secretory cell lineages (Basak et al., 2014; Buczacki et al., 2013; van Es et al., 2012) or triggering mature quiescent Paneth cells to reacquire stem cell properties in situations of damage and repair (Roth et al., 2012). Either mechanism could account for repopulation in our experiments.
Notably, FZD7 plays a non-redundant role in maintaining pluripotency of human embryonic stem cells (Fernandez et al., 2014) and might play a similar role in the intestinal stem cells. Our study demonstrates that intestinal stem cells that do not express Fzd7 have an inherent defect in Wnt/β-catenin signaling, which compromises stem cell function under conditions of stress. Fzd7 therefore might provide an avenue to specifically manipulate Wnt-driven processes in intestinal stem cells.

We previously reported that precursor cells expressing

We previously reported that precursor colony stimulating factor 1 receptor expressing the Myf5-Cre knockin driver give rise to a subset of white adipocytes in addition to brown adipocytes and skeletal muscle (Hung et al., 2014; Sanchez-Gurmaches and Guertin, 2014; Sanchez-Gurmaches et al., 2012; Tallquist et al., 2000). In young C57Bl/6 mice, nearly all of the anterior subcutaneous and retroperitoneal visceral white adipocytes are labeled with Myf5-Cre whereas all posterior subcutaneous white adipocytes and all mesenteric and perigonadal visceral white adipocytes are Myf5-Cre negative (Sanchez-Gurmaches and Guertin, 2014). Consistently, Myf5-Cre also labels many, but not all, of the APCs in the anterior subcutaneous and retroperitoneal depots. A similar pattern is recapitulated with Pax3-Cre in most depots with some notable exceptions including additional labeling of many perigonadal white adipocytes in male, but not female, mice (Sanchez-Gurmaches and Guertin, 2014). These findings reveal an unexpected degree of heterogeneity with respect to the Myf5-Cre and Pax3-Cre labeling in white fat; however, the utility of using the Myf5-Cre and Pax3-Cre drivers for in vivo genetic manipulation of white adipocyte precursors is limited because they neither uniformly nor selectively target any adipocyte class but rather appear to label adipocytes based on their anatomical location (i.e., dorsal-anterior) and in addition target some non-adipose tissues.
To identify Cre drivers that might be useful for targeting different adipocytes populations, we scanned the literature for well-characterized Cre drivers that have propensity to label adipocyte precursors and tested their ability to activate a reporter in different fat depots. One candidate was paired related homeobox transcription factor 1 Cre (Prx1-Cre) because it reportedly expresses in the mesenchymal precursors of the limbs and head (Logan et al., 2002), was recently reported to express in precursors of both brown and white adipocytes (Calo et al., 2010), and because PRX1 itself reportedly functions in adipogenesis (Du et al., 2013). A very recent report by Krueger and colleagues also indicates that Prx1-Cre labels inguinal subcutaneous adipocyte precursors (Krueger et al., 2014). Here, we provide a detailed analysis of Prx1-Cre labeling in fat and other tissues and show that Prx1-Cre labeling is largely restricted among the fat depots to the precursor and mature subcutaneous white adipocytes. Furthermore, we show that Prx1-Cre labeling of subcutaneous WAT is stable and that Prx1-Cre also labels brite/beige adipocytes in subcutaneous WAT. Finally, the utility of using Prx1-Cre and other Cre drivers for lineage tracing, cell marking, and conditional gene deletion in adipose tissue is discussed.


Recently, Krueger et al. (2014) reported that Prx1-Cre labels inguinal (posterior subcutaneous) white adipocyte precursors. Taken together, our studies provide complimentary evidence that, in the mouse, Prx1-Cre transgene activity labels adipocyte progenitors of subcutaneous white adipocytes. In further support, a recent study using Prx1-Cre to delete Ptpn11, which encodes SHP-2 phosphatase, notes that subcutaneous adipose tissue is absent in Prx1-Cre;Ptpn11 floxed mice (Lapinski et al., 2013), which is consistent with another recent study reporting a role for SHP-2 in adipogenesis (He et al., 2013). Thus, Prx1-Cre may be useful for conditional gene knockout studies in scWAT, especially when targeting genes with adipocyte-specific functions. However, it is essential that any study employing Prx1-Cre to target subcutaneous adipocytes pay careful attention to osteoblasts and chondrocytes (Logan et al., 2002). Of note, another recent study finds that Wt1-Cre selectively labels progenitors of visceral white adipocytes (Chau et al., 2014). Thus, comparative studies using Wt1-Cre and Prx1-Cre, when used to manipulate genes encoding fat-specific regulators, could provide valuable insight about visceral versus subcutaneous fat function.

br Discussion Our study provides insights into the mechanisms

Our study provides insights into the mechanisms underlying the loss of regenerative potential and increasing fibrogenesis with age in DMD. We report that a proportion of specialized cells, which are critical for muscle regeneration, cannot maintain their identifying functions in aged dystrophic muscle of humans and mice, and acquire instead the capacity to produce matrix proteins. This is in agreement with a recent report from the Rando group (Biressi et al., 2014). Fibrogenesis in dystrophic muscle does not appear to be an all-or-nothing but rather a partial transition, as most SAG share original as well as new fibroblastic traits, resembling the partial EMTs occurring in carcinosarcomas and fibrotic liver (Nieto, 2013; Sarrió et al., 2008; Zeisberg et al., 2007). This cellular plasticity, however, hampers muscle tissue repair potential. Mechanistically, we show that the loss of cell identity toward fibrogenesis in dystrophic muscle may involve the acquisition of mesenchymal traits, triggered by increasing TGFβ as disease progresses. This link between skeletal muscle fibrogenesis and mesenchymal-like transitional states, resulting in the loss of regenerative potential, was a striking finding. Because adipose tissue also accumulates in muscle of DMD patients, we postulated that the loss of identity of specialized cells concomitantly with the acquisition of mesenchymal-like (multipotent) characteristics might be a general feature for fibrogenesis as for adipogenesis in DMD, and by extension in other pathological conditions coursing with high TGFβ signaling and tissue damage. Indeed, TGFβ2- or BMP4-induced EndMT into multipotent stem-like cells was proposed to be the origin of heterotopic cartilage and bone in individuals with fibrodysplasia ossificans progressiva (FOP) lesions (Medici et al., 2010). A distinct muscle-resident multipotent progenitor additionally was proposed as an origin for FOP, based on its osteogenic potential in response to BMP2 (Wosczyna et al., 2012).
Recent studies have shown that, in addition to fibroblasts, tissue-resident FAPs and perivascular cells, which express PDGFRα, can differentiate to a fibroblastic fate and contribute to collagen accumulation in acutely injured muscle (Dulauroy et al., 2012; Joe et al., 2010; Uezumi et al., 2010). FAPs also accumulate in young dystrophic muscle prior to maximal fibrosis (Uezumi et al., 2011), being the main source of the fibrogenic progeny leading to collagen production at early dystrophy stages. Our results demonstrate that at advanced DMD stages there are additional reservoirs of fibrogenic cells derived from myogenic, endothelial, and hematopoietic cells. However, unlike organs like kidney, where a great proportion of the fibrotic cells arise from the bone marrow (and to a lesser extent from EndMT or EMT) (LeBleu et al., 2013), in dystrophic skeletal muscle, the net contribution of these cells to the actual population of collagen-producing cells (the key cells for fibrosis development) is modest. Instead, our results reveal that it is the loss of myogenic and endothelial cells’ biochemical and phenotypic identities, through plastic mesenchymal transitions (characterized by the expression of PDGFRα), that causes severe deficits in myogenesis and angiogenesis, thus exacerbating the regenerative impairment in dystrophic muscle (see scheme in Figure 1). Therapeutically, a unifying model of loss of cell fate and acquisition of mesenchymal traits centered on the PDGFRα-expressing cell could be envisioned. How to restrict deleterious PDGFRα-dependent functions while preserving the beneficial ones is likely to be more challenging.

Experimental Procedures

We are indebted to V. Ruiz-Bonilla, V. Lukesova, S. Gutarra, M. Raya, B. Ampudia, and members of the Cell Biology Group for their contributions to this study. J. Martín-Caballero (PRBB Animal Facility), O. Fornas (CRG/UPF FACS unit), and CRG Genomic Unit. We also thank M. Reyes and N. Ieronimakis for help in isolation of muscle endothelial cells, S. Biressi and T. Rando for the generous offer to provide samples and for information exchange, C. Keller for Pax7-Cre lines, R.H. Adams and J.L de la Pompa for Ve-CadER-Cre and Tie2-Cre lines, D. Brenner for ColI-GFP reporter mice, and D. Medici for advice on cell plasticity studies. The authors acknowledge funding from the Ministry of Economy and Competitiveness (MINECO)-Spain (SAF2012-38547, PI13/02512, and PLE2009-0124), Association Française Myopathies (AFM), E-Rare, Fundació Marató TV3, Muscular Dystrophy Association (MDA), European Commission Research and Innovation funding EU-FP7 (Myoage, Optistem, and Endostem), and Duchenne PP-NL. P.P. and Y.K. were partly supported by postdoctoral fellowships from AFM.