br Materials and methods br Results

Materials and methods

Results

Discussion
The roles of endogenous stem/progenitor Flavopiridol hydrochloride in adult pancreatic tissue homeostasis, acute/chronic injury response and tumor initiation remain uncertain. However, it is now clear that a subset of pancreatic duct cells are capable of initiating long-term self-renewing organoid cultures with effectively unlimited potential for expansion in culture. The M+133+26− subsets of both the pancreas and liver of adult mice contain the only cells with this capability. Lgr5, which is a marker for self-renewing stem cells in the colon, small intestine and stomach only becomes expressed after injury in the adult liver and pancreas (Barker et al., 2007, 2010; Huch et al., 2013a,b). Hence, in these latter tissues Lgr5 is only a marker for activated progenitors and cannot be used to prospectively identify clonogenic cells in normal, uninjured tissue. Our study is the first to report the prospective purification of “inactive” pancreatic and liver mouse organoid forming progenitors. We applied population expression profiling with RNA-seq and single cell analyses to determine the transcriptomes and transcriptional heterogeneity of these important populations. The ability to purify both inactive and injury-activated progenitors will permit the study of the molecular circuitry underlying progenitor activation in the future.
Although heterogeneity of pancreatic ducts has long been known based on morphological criteria, we demonstrate here for the first time that pancreatic duct cells are heterogeneous in terms of clonogenic potential. Thus it is appropriate to distinguish between clonogenic and non-clonogenic ducts. The surface marker combinations used herein do not permit the unambiguous identification of these duct subsets in histological sections. Hence, we currently do not know whether clonogenic and non-clonogenic ducts reside in anatomically distinct locations or whether they are intermingled and lie adjacent to each other. Our RNA-seq analysis represents a list of distinctive markers that could be used to localize the progenitors in tissue sections and generate lineage-tracing tools in the future. The observation of high Sox9 expression in only a small subset of M+133+26− is interesting, and it is tempting to speculate that this property is predictive of their organoid-forming capacity. We note that heterogeneity of Sox9 at the protein level has recently been reported in epithelial tissues. Ramalingam et al. found that intestinal epithelial cells express Sox9 at distinct levels, and that only the highest expressing subpopulation had progenitor activity (Ramalingam et al., 2012).
We have recently shown that gall bladder derived epithelial cells can be reprogrammed to become endocrine-like and express insulin using a combination of Pdx1, Ngn3 and MafA (Hickey et al., 2013). Others have shown that biliary duct cells in the liver can be reprogrammed in vivo and cure diabetes in mice (Akinci et al., 2012). Here we demonstrate that pancreatic duct cells can similarly be reprogrammed even after massive expansion in organoid culture. The expandability of the pancreatic progenitors makes them a potential target for generating transplantable beta cells. Ducts and acinar cells are routine by-products of islet isolation procedures using in clinical transplantation (Baeyens and Bouwens, 2008). Recently, the laboratory of S. Kim has shown that human ducts can also be reprogrammed to become functional beta-like cells (Lee et al., 2013). Therefore, the progenitor population described here may have potential utility in beta cell replacement therapy for diabetes.
The following are the supplementary data related to this article.

Acknowledgments

Introduction
Primary cilia (non-motile) are cellular extensions extending from most growth arrested mammalian cells and are responsible for coordinating responses to extracellular conditions (Basten and Giles, 2013; Christensen et al., 2008; Quarmby and Parker, 2005; Satir et al., 2010). Primary cilia sense the extra-cellular environment by chemosensation and mechanotransduction, transducing this information through signaling cascades, allowing cells to respond to environmental stimuli (Avasthi and Marshall, 2012; Davenport and Yoder, 2005; Han et al., 2008; Malone et al., 2007; Oh and Katsanis, 2012). The primary cilium is comprised of two main parts: 1) a basal body, consisting of modified centrioles and 2) a ciliary axoneme, comprised of a ring of 9 microtubule doublets bound by the cell membrane. As sensory organs primary cilia [9(+0)] lack the central structural microtubule doublet (+2) and dynein arms required for motile cilia [9(+2)] (Basten and Giles, 2013; Takeda and Narita, 2012). Importantly, defects in primary cilia have been found to be the causative agent behind many human diseases or developmental defects (Nigg and Raff, 2009; Oh and Katsanis, 2012; Pan et al., 2005; Schmidt et al., 2012; van Reeuwijk et al., 2011; Weatherbee et al., 2009; Zalli et al., 2012).

br Materials and methods br Results

Materials and methods

Results

Discussion
The roles of endogenous stem/progenitor Flavopiridol hydrochloride in adult pancreatic tissue homeostasis, acute/chronic injury response and tumor initiation remain uncertain. However, it is now clear that a subset of pancreatic duct cells are capable of initiating long-term self-renewing organoid cultures with effectively unlimited potential for expansion in culture. The M+133+26− subsets of both the pancreas and liver of adult mice contain the only cells with this capability. Lgr5, which is a marker for self-renewing stem cells in the colon, small intestine and stomach only becomes expressed after injury in the adult liver and pancreas (Barker et al., 2007, 2010; Huch et al., 2013a,b). Hence, in these latter tissues Lgr5 is only a marker for activated progenitors and cannot be used to prospectively identify clonogenic cells in normal, uninjured tissue. Our study is the first to report the prospective purification of “inactive” pancreatic and liver mouse organoid forming progenitors. We applied population expression profiling with RNA-seq and single cell analyses to determine the transcriptomes and transcriptional heterogeneity of these important populations. The ability to purify both inactive and injury-activated progenitors will permit the study of the molecular circuitry underlying progenitor activation in the future.
Although heterogeneity of pancreatic ducts has long been known based on morphological criteria, we demonstrate here for the first time that pancreatic duct cells are heterogeneous in terms of clonogenic potential. Thus it is appropriate to distinguish between clonogenic and non-clonogenic ducts. The surface marker combinations used herein do not permit the unambiguous identification of these duct subsets in histological sections. Hence, we currently do not know whether clonogenic and non-clonogenic ducts reside in anatomically distinct locations or whether they are intermingled and lie adjacent to each other. Our RNA-seq analysis represents a list of distinctive markers that could be used to localize the progenitors in tissue sections and generate lineage-tracing tools in the future. The observation of high Sox9 expression in only a small subset of M+133+26− is interesting, and it is tempting to speculate that this property is predictive of their organoid-forming capacity. We note that heterogeneity of Sox9 at the protein level has recently been reported in epithelial tissues. Ramalingam et al. found that intestinal epithelial cells express Sox9 at distinct levels, and that only the highest expressing subpopulation had progenitor activity (Ramalingam et al., 2012).
We have recently shown that gall bladder derived epithelial cells can be reprogrammed to become endocrine-like and express insulin using a combination of Pdx1, Ngn3 and MafA (Hickey et al., 2013). Others have shown that biliary duct cells in the liver can be reprogrammed in vivo and cure diabetes in mice (Akinci et al., 2012). Here we demonstrate that pancreatic duct cells can similarly be reprogrammed even after massive expansion in organoid culture. The expandability of the pancreatic progenitors makes them a potential target for generating transplantable beta cells. Ducts and acinar cells are routine by-products of islet isolation procedures using in clinical transplantation (Baeyens and Bouwens, 2008). Recently, the laboratory of S. Kim has shown that human ducts can also be reprogrammed to become functional beta-like cells (Lee et al., 2013). Therefore, the progenitor population described here may have potential utility in beta cell replacement therapy for diabetes.
The following are the supplementary data related to this article.

Acknowledgments

Introduction
Primary cilia (non-motile) are cellular extensions extending from most growth arrested mammalian cells and are responsible for coordinating responses to extracellular conditions (Basten and Giles, 2013; Christensen et al., 2008; Quarmby and Parker, 2005; Satir et al., 2010). Primary cilia sense the extra-cellular environment by chemosensation and mechanotransduction, transducing this information through signaling cascades, allowing cells to respond to environmental stimuli (Avasthi and Marshall, 2012; Davenport and Yoder, 2005; Han et al., 2008; Malone et al., 2007; Oh and Katsanis, 2012). The primary cilium is comprised of two main parts: 1) a basal body, consisting of modified centrioles and 2) a ciliary axoneme, comprised of a ring of 9 microtubule doublets bound by the cell membrane. As sensory organs primary cilia [9(+0)] lack the central structural microtubule doublet (+2) and dynein arms required for motile cilia [9(+2)] (Basten and Giles, 2013; Takeda and Narita, 2012). Importantly, defects in primary cilia have been found to be the causative agent behind many human diseases or developmental defects (Nigg and Raff, 2009; Oh and Katsanis, 2012; Pan et al., 2005; Schmidt et al., 2012; van Reeuwijk et al., 2011; Weatherbee et al., 2009; Zalli et al., 2012).

br Summary and conclusions br

Summary and conclusions

Introduction
Historically, the foundations of the concept that the heart is a static organ incapable of regeneration were established in the mid-1920s. A significant publication in 1925 claimed that mitotic figures are not detectable in human cardiomyocytes (Karsner et al., 1925), which were considered as cells irreversibly withdrawn from the cell cycle. This work challenged numerous studies published from 1850 to 1911 in which the general belief was that cardiac hypertrophy was the consequence of hyperplasia and hypertrophy of existing cardiomyocytes (for review see Anversa and Kajstura, 1998). The 1925 report introduced the notion that the heart is a terminally differentiated post-mitotic organ.
The conclusion that new cardiomyocytes cannot be formed in the human heart was mostly dictated by difficulties in identifying mitotic nuclei. The conviction that the pool of myocytes present at birth is irreplaceable during the lifespan of the organism gained further support from a series of autoradiographic studies conducted in the late 1960s and early 1970s (Anversa and Kajstura, 1998; Soonpaa and Field, 1998). The degree of DNA synthesis in myocyte nuclei was negligible, and this observation, together with the inability to distinguish mitotic images in cardiomyocytes, reiterated the theory that the adult heart is composed of a homogenous population of parenchymal cells that are in a permanent state of growth arrest.
However, quantitative measurements of myocyte volume and number, performed in human hearts collected from patients who died as a result of decompensated cardiac hypertrophy and chronic heart failure, began to challenge this concept of myocardial biology. In the late 1940s and early 1950s, Linzbach documented that, in the presence of a heart weight equal to or greater than 500g, myocyte proliferation represented the predominant mechanism of the increase in cardiac muscle mass (Linzbach, 1947, 1960). These results were confirmed several years later (Adler and Friedburg, 1986; Astorri et al., 1971, 1977). In all cases, hearts weighing 500g or more were characterized by a striking increase in myocyte number that was more prominent than cellular hypertrophy; this order Wnt-C59 involved the left and right ventricular myocardium.
An inherent inconsistency became apparent. If we assume that cardiomyocytes lack the ability to reenter the cell cycle and replicate, differences in myocyte size would be expected to reflect comparable differences in the size of the organ. However, changes in heart weight and cardiomyocyte volume rarely coincide challenging the notion that the number of myocytes is an entity that remains constant throughout the organ lifespan. This discrepancy has been reported frequently with postnatal maturation, myocardial aging and cardiac diseases (Anversa and Kajstura, 1998; Anversa et al., 1998). Changes in myocyte number are the consequence of two interrelated mechanisms, myocyte death and myocyte formation. This rather simple biological principle is often ignored; the plasticity of the myocardium cannot be equated to myocyte hypertrophy only.
The critical interaction of cell death and cell renewal is not unique to the heart. Organ mass in prenatal and postnatal life is determined by the balance between cell death and cell division, which regulate the number of parenchymal cells within the tissue (Hipfner and Cohen, 2004). With various diseases, cell loss may be compensated by an increase in size of the surviving cells, although this response may become rapidly maladaptive in view of the difficulty of hypertrophied cells to perform efficiently their specialized function (Gomer, 2001).
Postnatal cardiac development, endurance exercise training, and pregnancy are typical examples of physiological cardiac hypertrophy (Dorn, 2007; Hill and Olson, 2008). The rapid expansion in myocardial mass after birth in mammals involves both an increase in size and number of cardiomyocytes, but the growth of the coronary vasculature markedly exceeds the growth of the myocyte compartment (Anversa and Olivetti, 2002; Rakusan et al., 1994). It is difficult to compare the dramatic increase in heart weight that occurs postnatally with the relatively modest degree of cardiac hypertrophy promoted by dynamic exercise. Additionally, there is little information concerning the cellular basis of exercise- and pregnancy-induced myocardial hypertrophy. Similarly, the mechanisms implicated in the regression of cardiac hypertrophy with loss of physical conditioning, or following delivery, have not been determined. Whether new myocytes are formed with endurance exercise and pregnancy and whether myocyte loss, myocyte atrophy, or both, contribute to the restoration of myocardial mass with cessation of exercise and pregnancy are unknown. Thus far, the only conclusion that can be reached is that preservation of myocardial structure characterizes postnatal development, moderate endurance training, pregnancy, and the early phases of increased pressure and volume loading on the adult heart. This balanced “physiological” response, however, is temporary, and aging, strenuous exercise, and sustained workload lead to the structural and functional manifestations of “pathological” hypertrophy, pointing to “time” as the critical determinant of the transition from physiological to pathological cardiac hypertrophy (Dorn, 2007).

br Material methods br Results br

Material & methods

Results

Discussion
Several LSC culture systems have been developed to re-create the LSC niche in vitro and to avoid the cross-contamination with non-human feeder cycloastragenol (Sharma et al., 2012; Xie et al., 2012; Chen et al., 2013; Mei et al., 2014). It has been suggested that feeder cells are required to maintain a stratified epithelial sheet and to regenerate a sufficient number of progenitor cells in culture (Miyashita et al., 2008). In the present study, LSCs were cultured with human BMSCs as feeder cells cycloastragenol in different systems to determine whether these BMSCs were a suitable replacement of mouse 3T3 feeder cells. To compare the quality of different culture systems, we evaluated several parameters such as cell morphology, cell growth success rate, PD rate, the proportion of stem/progenitor cells, and the proportion of mature corneal epithelial cells.
Based on the quantifiable measures that we evaluated, we determined that single LSCs could not be efficiently cultured on BMSCs in each culture method tested since differentiation in these cultures was increased (p63α data not shown). This finding is consistent with a previous observation (Omoto et al., 2009). LSCs cultured in the form of cell clusters achieve a higher expansion efficiency of the progenitor cell population (Kawakita et al., 2009; Gonzalez and Deng, 2013). In contrast, LSC clusters cultured in the 2D system produced cell outgrowths with a very heterogeneous morphology and a significantly low number of p63αbright cells; the presence of large differentiated cells at the center of the outgrowth, away from the feeder cells, suggested that nutrients secreted by the BMSC feeder cells could not reach this central area. In addition, the cell growth success rate was significantly lower in the 2D culture methods regardless of whether LSCs were seeded as single cells or as cell clusters. In the cultures of successful growth, the size of cell outgrowths was small and the cell morphology was consistent with that of differentiated epithelial cells at the center of the colony. These observations suggest that BMSCs can partially support the growth of LSCs in a 2D culture system.
Apart from the insufficient nutrient supply, the 2D method has other disadvantages that need to be overcome if cells produced in this system are used in a clinical setting (Mei et al., 2014). First, both LSCs and feeder cells compete for the growth surface; LSCs push away the feeder cells as they grow, and this competition for the growth area might lead to a decrease in the number of feeder cells and an insufficient nutrient supply. Second, cross-contamination with the feeder cells is possible because both types of cells are grown in direct contact. To avoid these shortcomings in the 2D culture method, the 3D method was developed; this method has been shown to support the LSC phenotype and increase the expansion rate when mouse 3T3 feeder cells are used (Mei et al., 2014).
LSCs grown in the 3D CC-BM system demonstrated the most homogeneous limbal epithelial morphology. The cultured epithelial cells were small and cuboidal in shape with very tight cell-to-cell junctions. The cell expansion rate and the percentage of K14+ and p63αbright cells in the 3D CC-BM system were equivalent to the control. Although p63α is expressed in both TA and stem cells, cells expressing high levels of p63α are usually located at the basal layer of the limbus where LSCs reside. Moreover, the only known predictor of clinical outcome is the percentage of the p63αbright cells (Rama et al., 2010).
The proportion of K12+ cells in the 3D system was comparable to the control. The differences did not reach statistical significance and such low levels were clinically negligible. Our finding supports the hypothesis that the 3D culture system is better than the standard 2D system in mimicking the in vivo spatial environment of LSCs (Mei et al., 2014). In this 3D culture system, LSCs are evenly distributed and in close contact with BMSCs; this arrangement allows a more homogeneous niche support that is not present in the direct method of culture. Moreover, cross-contamination with feeder cells is avoided in this 3D method.

br Materials and methods br

Materials and methods

Acknowledgements
Mef2c-AHF-Cre mice were generously provided by Dr. Brian L. Black. We thank Mamen Martín and Sandra Rodríguez (Molecular Cytogenetics Unit, CNIO) for technical advice; and the Imaging Core Facility for the confocal analysis (CIMA, University of Navarra). We thank the “Pluripotency Group” at the Department of Cell Therapy (FIMA) for the daily input and support, especially to Xabier L. Aranguren, Juan R. Rodriguez, Adrian Ruiz-Villalba and Beatriz Pelacho. This work was supported by the “Ramón y Cajal” State Program, Ministry of Economy and Competitiveness (MINECO, RYC-2012-10981) to XCV; the “Retos de la Sociedad” State Program, MINECO (SAF2013-46142-R) to XCV; “Promoción del Talento y su Empleabilidad” State Program, MINECO (BES-2014-069226) to JLA; “Red de Terapia Celular”, Ministry of Health, Social Services and Equality-Institute of Health Carlos III (TERCEL ISCIII-RETIC RD12/0019/0031) to FP.

Resource table

Resource details
A total of 9 embryos from in vitro phenylephrine hydrochloride (IVF) and 97 embryos from preimplantation genetic diagnosis (PGD) were donated for research in accordance with the legal requirements of the country of origin by donors included in PGD program at Unidad de Gestión Clínica de Genética, Reproducción y Medicina Fetal (UGC) from University Hospital Virgen del Rocío. The donors gave written informed consent (Cortes JL et al., 2007 and Fernández et al., 2014).
Human embryos were thawed using Thaw Kit 1TM de Vitrolife® and cultured using G2 medium (GIII series, Vitrolife®), inner cell mass (ICM) was mechanically isolated under stereomicroscope using 2 insulin syringe (25G) at “hatching blastocyst” stage (Ström et al., 2007) and plated onto mitomycin-C inactivated human newborn foreskin fibroblasts (hFFs), the resulting colonies displayed the typical morphology of hESCs (Fig. 1A) and are positive to alkaline phosphatase staining (Fig. 1B).
The analysis of pluripotent markers was evaluated by RT–PCR, immunofluorescence and flow cytometry. Undifferentiated HVR1, HVR2 and HVR3 cells expressed Oct4, Sox2, Nanog and Telomerase (TERT) detected by RT–PCR (Fig. 1C), are positive to OCT4, SSEA4, TRA-1-60 and TRA-1-81 proteins assessed by immunostaining (Fig. 1D) and more than 90% of cells were positive to SEEA4 (Fig. 1E).
The ability to differentiate into three germ layers was demonstrated by in vitro differentiation of embryoid bodies (EBs) and by in vivo teratoma formation after cells implantation into immune-deficient mice. Differentiated cells from HVR1, HVR2 and HVR3 expressed neuronal class III β-tubulin ectoderm marker (Tuj1), human cardiac troponin T mesoderm marker (cTnT) and human α-Fetoprotein endoderm marker (AFP) (Fig. 2A), while hematoxylin and eosin (H&E) and alcian blue contrastained sections from the teratomas indicated that the cell lines HVR1, HVR2 and HVR3 differentiated in vivo to all three germ layers (Fig. 2B).
The karyotype analysis showed a stable karyotype 46,XY for HVR1 (Fig. 3A) and 46,XX for HVR3 (Fig. 3B), however HVR2 presented a karyotype with chromosome translocation 46,XY, t(9;15)(q34.3;q14) (Fig. 3C); this translocation has been also detected with FISH analysis (Fig. 3D).

Materials and methods

Verification and authentication

Author disclosure statement

Acknowledgments
This work was supported by a non-profit Foundation ‘Fundación Progreso y Salud’ of the Andalusian Regional Ministry of Health; Consejería de Economía y Conocimiento, Junta de Andalucía and Fondo Europeo de Desarrollo Regional (FEDER) (TCMR0021/06 and PI246-2008). Authors are supported by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER) (RD12/0019/0028 and RD012/0036/0017; PI10/00964; PI11/02923 and PI14/01015); the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). Support from FSED and FAID allowed access to databanks. CIBERDEM and CIBERER are initiatives of the Instituto de Salud Carlos III.

By studying the relationships of these subpopulations to

By studying the relationships of these subpopulations to each other, it was found that GMPs are able to create neutrophils but unexpectedly lack the potential to form eosinophils and basophils. Furthermore, and against the prevailing assumption, the GMPs were found to be derivatives of the same branch of hematopoiesis as the lymphocytes, pointing toward altered lineage relationships in human hematopoiesis (Görgens et al., 2013b). Accordingly, we recently proposed a revised model of human hematopoiesis (Görgens et al., 2013a, 2013b). Another outcome of this study was the observation that under the conditions used, MPPs cannot self-renew in vitro; following their first in vitro cell division, they apparently create CD133-positive LMPPs and CD133-negative EMPs, maybe by means of ACD (Görgens et al., 2013a, 2013b). Enforcing assumed roles of ACDs in this lineage-separation process, asymmetric segregation of CD133 molecules was observed in a proportion of dividing CD34+ cells at the intracellular level (Fonseca et al., 2008). In contrast, and independent of its intracellular distribution, the extracellular component of CD133 appeared to be symmetrically distributed on all dividing CD34+ cells (Beckmann et al., 2007; Fonseca et al., 2008).
In addition to the cell-fate analyses and ACD studies, we compared the distribution of CD133 at the subcellular level on freshly isolated and cultured HSPCs. Upon cultivation, HSPCs adopt a polarized morphology, forming a leading edge at the front and a leukocyte-specific structure, the uropod, at the rear (Giebel et al., 2004; Rajendran et al., 2009). While CD133 showed a rather random appearance on freshly isolated HSPCs, it redistributes to the uropod tips in cultured HSPCs (Giebel et al., 2004; Görgens et al., 2012). In our studies, we learned that the CD133 epitopes that are recognized by commonly used anti-CD133 Decitabine (AC133 and AC141) are sensitive to paraformaldehyde fixation (Giebel et al., 2004). Both of these antibodies have been reported to recognize spatially distinct, glycosylation-dependent residues on the extracellular CD133 loops (Bidlingmaier et al., 2008; Miraglia et al., 1997). Recently, a novel monoclonal anti-CD133 antibody, the HC7 antibody, has been described, which specifically recognizes glycosylation-independent protein residues of both extracellular loops of CD133 (Swaminathan et al., 2010). This antibody had been successfully used in a variety of applications including western blot, immunofluorescence, flow cytometry, and immunohistochemistry using cancer cell lines (Swaminathan et al., 2010).
To test for the usefulness of the HC7 antibody in hematopoietic stem cell research, we comprehensively compared the presence and subcellular distribution of HC7 and glycosylation-dependent CD133 epitopes on freshly isolated and cultured UCB-derived CD133+CD34+ HSPCs. Furthermore, we tested whether binding of the HC7 or the AC133 antibody exerts any functional impact on the biology of human HSPCs, including their potential to home and engraft into immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After showing that neither AC133 nor HC7 antibodies recognizably alter biological features of human CD133+CD34+ cells and that both antibodies allow detection of living, asymmetrically dividing HSPCs, we compared the ACD rates in MPP-, LMPP-, and GMP-enriched fractions of HC7-stained cells and analyzed MPP daughter cells at a single-cell level, both phenotypically and functionally.

Results

Discussion
By using the HC7 anti-CD133 antibody, we demonstrate that almost all human HSPCs of the MPP fraction with confirmed lymphomyeloid and erythromyeloid developmental potential perform ACDs in vitro to create a lymphomyeloid CD34+CD133+CD45RA+ and a erythromyeloid CD34+CD133−CD45RA− daughter cell. In this context, we first comprehensively tested for the usability of the HC7 anti-CD133 antibody in human hematopoietic stem cell research. Side-by-side comparison of HC7 and the classical AC133 anti-CD133 antibody revealed that on fixed mitotic HSPCs, HC7, in contrast to AC133, allows detection of asymmetric CD133 distribution. Otherwise, no difference was detected regarding either their epitope expression on human HSPC subsets or their subcellular distribution on polarized or living mitotic HSPCs. Therefore, we conclude that the different appearance on fixed mitotic cells is an artifact caused by paraformaldehyde fixation rather than a difference in the HC7 and AC133 epitope distribution on HSPCs. Side-by-side comparison of HC7 or AC133 binding to human CD133+ HSPCs did not reveal a recognizable impact of any of these antibodies on the cell biological features of HSPCs, including their homing and repopulation capabilities in NOD/SCID mice. Thus, we consider the binding of both antibodies as functional neutral and conclude that they provide valuable tools for live-cell analyses of human CD133+ HSPCs.

The comparison of the modeled and the

The comparison of the modeled and the experimental images (Fig. 6) showed that the experimental images for small QDs for g = 220 (see Fig. 6 (d)) match the simulations in moiré period and the number of moiré fringes with a 6% lattice mismatch (Fig. 6 (a)). This means that small QDs consist of pure indium antimonide and have no arsenic inclusions (or a small amount of it).
For a 3 and a 6% mismatch distinctive ‘ticks’ (Fig. 6 (b) and (c)) can be observed in simulated AN-2728 images with the diffraction vector g = 400 and 0–40; the same ‘ticks’ appear in experimental TEM images of large QDs (Fig. 6 (e) and (f)). This proves that misfit defects are present on the quantum dot/substrate interface for larger QDs.

Conclusions

Acknowledgments

Introduction
This so-called laser ignition device (LID) was designed by the Keldysh Research Centre (Moscow) together with the Energomash R&D complex (Khimki, Moscow oblast). Energomash has been bench-testing the LIDs since 2011 [1].
Developing and integrating new rocket engine elements such as LIDs is going to entail copious bench and in situ tests requiring much time and expense. Furthermore, the tested system itself is rather complex, with over 15 adjustable installation parameters, about 10 observables, and about a hundred accompanying measurements. Due to these difficulties, only 34 tests have been conducted from 2011 to 2013.
To save time and material costs, specifically, to reduce the number of in situ tests, it seems prudent to use mathematical methods and algorithms to predict the responses to inputs of the system as a whole and of the system\’s individual elements.

The advantages of a trainable neural network
In the present paper, the predictive model of the system behavior has been constructed using the trainable neural network described in [2] that has the following advantages.
Firstly, each of the network\’s computing elements (neurons) is simple. A neuron is a weighted adder with the output defined as
where is an i-th input of a neuron, is a synaptic weight of an i-th input connection, f is an activation function.
The input x0 that is a fixed input signal +1 and its matching weight w0 is called a neuron bias.

The stages of building a neural network prediction model
Let us list the stages of building this model.

Predicting the gas temperature in the orifice plate
Each set includes the pair of an input vector (a column from the input parameter matrix X) and an output value (a corresponding value from the output parameter vector Y).
The network was trained using the error back-propagation algorithm that is a modified gradient descent method [2]; weighting coefficient increments for each iteration were found by the formula
where is the weighting coefficient of the connection between the i-th neuron of layer k – 1 with the j-th neuron of layer k; η is a training rate coefficient, 0 < η < 1. Fig. 3 shows regressograms of the training the neural network, plotted using the NNtool box from the MATLAB package. Table 4 lists the results of analyzing plots 2 in Fig. 3 for a synthesized neural network, including the ratios of actual and target values. For R = 1 there is an exact linear relationship between the neural network output and the target value, while if R is close to zero, there is no linear relationship between these values [9]. The results obtained for the test set (the three experimental implementations that were not used in training) can be seen in Fig. 4 and Table 5.
Conclusion
We should specifically stress that it is possible to broaden the factor space and, as a result, improve the constructed model, with more in situ tests (including failed ones).

Introduction
A unique complex of methods of mathematical modeling, simulation and enhancement that are potentially applicable to various types of pipeline systems has been developed by the researchers of the Melentiev Energy Systems Institute as a part of the Institute\’s studies in the theory of hydraulic circuits [1–3]. However, as ready-to-use software simulation packages are designed, many of them are essentially the same, and inevitably require much time and resources to be adapted for practical use.

Future studies of human astrocyte response to inflammation require reliable

Future studies of human astrocyte response to inflammation require reliable in vitro models. Human astrocytes can be cultured from fetal and adult biopsies; however, the availability of CNS human tissue is reduced and immunopanning is required to obtain pure astrocyte preparations (Zhang et al., 2016). The generation of astrocytes from induced pluripotent stem cells (iPSCs) has the advantage of obtaining access to astrocyte phenotypes and their effects on neuronal physiology from patients with neurodegenerative and neuropsychiatric diseases. However, the development of efficient protocols to generate astrocytes in a dish from stem cells is hindered by insufficient knowledge of astrocyte specification during development and in the adult CNS and by the lack of specific proteins expressed by astrocytes that can be used as markers (Molofsky et al., 2012). Recently, Zhang et al. (2016) identified human astrocyte-specific genes and differences in transcription between astrocyte precursors and mature astrocytes. Current available protocols focus on recapitulating the gliogenic switch observed during embryonic development (Chandrasekaran et al., 2016; Emdad et al., 2012; Krencik and Zhang, 2011; Roybon et al., 2013; Serio et al., 2013; Shaltouki et al., 2013; Tyzack et al., 2016). Human iPSCs (hiPSCs) or human embryonic stem cells (hESCs) are converted into neural progenitor cells (NPCs) and then patterned and switched to glial progenitor cells (GPCs) that eventually differentiate and mature into astrocytes. Recently, the conversion of mouse and human fibroblasts into astrocytes using small-molecule induction has been described (Tian et al., 2016). Despite the role that astrocytes play in neuroinflammation, few studies have reported the generation of pro-inflammatory astrocytes (Holmqvist et al., 2015; Roybon et al., 2013). In this study, we describe an efficient protocol to derive functional astrocytes from glial progenitors that show a rapid inflammatory response after stimulation with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α).

Results

Discussion
Along with microglia and other non-neural cells, astrocytes participate in the inflammatory response of the CNS to injury and disease by producing cytokines, chemokines, pro-oxidant molecules, and signaling factors (DiSabato et al., 2016; Sofroniew, 2015). Astrocytes also play an important role in maintaining buy galanin function homeostasis, and their dysfunction can trigger progression of neurodegenerative diseases (Chandrasekaran et al., 2016). Much of our current knowledge on astrocyte biology comes from the study of rodents. Recently it was shown that human astrocytes are intrinsically more complex than mouse astrocytes, and astrocytes express unique genes and respond differently to extracellular glutamate (Han et al., 2013; Oberheim et al., 2006, 2009; Zhang et al., 2016). Importantly, drugs that showed promising results in animal models have failed in human trials (Cavanaugh, 2014; Cummings et al., 2014; Rothstein, 2003; Waldmeier et al., 2006), possibly due to the different properties of human astrocytes. Therefore, the development of applicable and relevant human models for studying neurological disease mechanisms and testing drugs has become necessary. Here we report an efficient method for differentiating inflammation-responsive astrocytes from human iPSCs and ESCs.
A major breakthrough in the study of astrocytes was the description of a procedure to purify and culture astrocytes from neonatal rodent brains using serum-containing media (McCarthy and de Vellis, 1980). This protocol was used and modified for decades to culture not only rodent cells but also human astrocytes from fetal postmortem tissue (Ennas et al., 1992; Lee et al., 1993). Recently, new methods of astrocyte purification using immunopanning combined with serum-free culture have been described for adult rodent and human brains (Foo et al., 2011; Zhang et al., 2016). These methods are attractive because the selected astrocytes are functional and are expected to be more similar to their in vivo equivalents. However, these are invasive techniques; for example, the temporal lobe cortex tissue used by Zhang et al. (2016) was excised during surgery to expose the epileptic hippocampi, and the broad application of this technique to the study of human neurological disorders remains challenging. In addition, most neuroinflammatory, neurodegenerative, and psychiatric conditions are multigenic and poorly described genetically. Thus, cellular models that rely on the differentiation of astrocytes from iPSCs or other non-brain cell types are still the most accessible methods for in vitro disease modeling and can potentially allow for new discoveries. Indeed, astrocyte dysfunction was found in Costello and Down syndromes using iPSC-derived astrocytes uncovering new drug targets (Chen et al., 2014; Krencik et al., 2015).

Already in the early eighties it was

Already in the early eighties, it was recognized that total IgG in patients with rheumatoid arthritis (RA) contained less galactose and sialic Phos-tag at the non-reducing termini compared to healthy controls (Parekh et al., 1985). A difference in the glycosylation profile of total IgG has since been demonstrated in patients with various other auto-immune diseases when compared to healthy controls, including systemic lupus erythematosus, inflammatory bowel disease, myasthenic gravis, ankylosing spondylitis, primary Sjögren\’s syndrome, psoriatic arthritis and multiple sclerosis (Watson et al., 1999; Wuhrer et al., 2015a; Vuckovic et al., 2015; Trbojevic Akmacic et al., 2015; Selman et al., 2011). Furthermore, significant differences have been observed in the glycosylation profile of total IgG and specific auto-antibodies, such as anti-citrullinated protein antibodies (ACPA), anti-β2GP1 and anti-histone IgG (Scherer et al., 2010; Rombouts et al., 2015; Magorivska et al., 2016; Fickentscher et al., 2015). Finally, Fc glycosylation of auto-antibodies may change during disease development. For example, the glycosylation profile of ACPA changes prior to the onset of RA towards a more inflammation-associated phenotype (Rombouts et al., 2015; Ercan et al., 2010).
In patients with PR3-ANCA, it was previously shown with lectin assays that total IgG exhibits a lower degree of galactosylation when compared to healthy controls (Holland et al., 1760, 2002). In addition, the degree of sialylation of PR3-ANCA is lower during active disease compared to inactive disease (Espy et al., 2011). Recently, it has been shown with mass spectrometric IgG Fc glycosylation analysis that total IgG Fc of patients with severe AAV exhibits lower levels of galactosylation, sialylation and bisecting N-acetylglucosamine (GlcNAc) compared to healthy controls (Wuhrer et al., 2015b). This finding was more pronounced for PR3-ANCA compared to total IgG (Wuhrer et al., 2015b). Correlations were observed between the glycosylation profile of PR3-ANCA and several cytokine concentrations, suggesting that the glycosylation of ANCA may be driven by T-cell activation in an antigen-specific manner (Wuhrer et al., 2015b). Potential differences and similarities in the glycosylation profile of total IgG and antigen-specific IgG between patients with PR3-ANCA and patients with MPO-ANCA associated vasculitis have not yet been investigated.

Materials and Methods

Results

Discussion
While IgG with a low degree of galactosylation has repeatedly been found to be associated with pro-inflammatory autoimmune responses, the underlying mechanisms are still largely elusive (Bondt et al., 2013; Matsumoto et al., 2000; Albrecht et al., 2014; Rademacher et al., 1994). In addition, highly galactosylated IgG may confer anti-inflammatory activities through the association with the inhibiting receptor FcyRIIb and the C-type lectin-like receptor Dectin-1 in mice (Karsten et al., 2012). This latter pathway has been shown to block C5a effector functions in vitro and C5a-dependent inflammatory responses in animal mouse models (Karsten et al., 2012). This is highly relevant in AAV, since C5a plays a pivotal role in the pathophysiology (Wilde et al., 2011; Hilhorst et al., 2015). Mouse models have shown that the blocking of C5a or C5a receptor (C5aR) ameliorates anti-MPO induced necrotizing glomerulonephritis (Xiao et al., 2014; Huugen et al., 2007). The safety and efficacy in the treatment of non-life-threatening AAV with CCX168, a C5aR inhibitor, is currently being tested in a phase 2 study (EudraCT Number: 2011-001222-15). The addition of galactose to the glycan structure also no longer enables the interaction of MBL with IgG, and may thereby block the lectin pathway (Malhotra et al., 1995). The clinical relevance of the lectin pathway in AAV is questionable however, since mannose-binding lectin (MBL) deposition in the kidney was found in only a minority of patients and complement activation in AAV occurs predominantly via the alternative pathway (Hilhorst et al., 2015).

In the present issue of Lupinek et al address

In the present issue of , Lupinek et al. address by a different approach the limit of too high total IgE levels or body mass precluding the use of omalizumab in severe asthma (). They used IgEnio (Fresenius Medical Care, Bad Homburg, Germany) a device that consist of a extracorporeal immunoadsorber column for selective removal of IgE from patients\’ plasma by the use of ScFv12, a recombinant single-chain variable fragment obtained by that selectively removes IgE. Fifteen patients with seasonal allergic asthma and IgE levels up to 4344U/ml, largely exceeding the limit for omalizumab treatment, were randomly assigned to treatment with IgEnio (10 patients), based on 3cycles of immune adsorptions with a 4week interval, or to the control group (5 patients). The two groups had comparable total IgE levels. At the end of the last cycle, IgE immune adsorption depleted 86.2% of IgE and also depleted immune complexes IgE-omalizumab (generated by introducing omalizumab in patients\’ plasma). Also, a reduction of allergen skin test and basophil sensitivity, as well as of clinical symptoms, was observed. The treatment was well-tolerated. According to authors, IgE immune adsorption can be proposed in patients with severe pollen-induced asthma and in patients with severe asthma who cannot be treated to omalizumab because of too high IgE levels. The first suggestion must be confirmed by clinical trials on large populations of pollen-allergic patients, while the second could be an option to the future use of the new anti-IgE monoclonal glucose assay (). In fact, omalizumab has an acknowledged role in severe uncontrolled allergic asthma but is significantly restricted by the limit of too high IgE levels or body mass. It seems reasonable to expect that this barrier could be overcome by a pre-treatment with IgEnio, to allow to excluded patients to be admitted to omalizumab treatment.
Disclosure

In Tucker and colleagues report findings from their meta-synthesis of qualitative data from three systematic reviews to holistically assess barriers common to all aspects of the HIV care continuum, including linkage to care, medication adherence, and retention in care (). Although previous reviews have examined specific aspects of the continuum, the review by Tucker et al. is unique in its perspective of viewing the continuum as a whole. The review included 24 studies across low-, middle-, and high-income countries that qualitatively evaluated interventions involving operational changes to improve either linkage to care, retention, or adherence.
This review is timely as the World Health Organization released updated guidelines in 2015 on antiretroviral drugs for treating and preventing HIV infection (). The new guidelines outline a “test and treat” approach recommending that everyone living with HIV begin antiretroviral therapy immediately following diagnosis, regardless of CD4 count. As evidence for both the preventative and treatment benefits of early antiretroviral therapy (ART) are now well-established, () the guidelines mark an important shift in the field. However, such an approach will only be effective if people living with HIV are willing and able to get tested, seek care, adhere to medication, and remain in care long-term. The review by Tucker et al. highlights obstacles people face across all aspects of the care continuum—signaling potential pitfalls to a successful test and treat strategy.
The implementation challenges of providing ART to all people living with HIV include barriers at individual, community, health system, and structural levels (). Understanding and addressing these obstacles are critical to improving uptake, adherence, and retention in care. The seven cross-cutting themes identified by Tucker et al. mainly fall into two categories—structural and health systems barriers. Structural barriers identified include poverty, transportation, food insecurity, glucose assay and housing. The review also identified gender as an issue, with fewer men accessing treatment than women. This can also be interpreted as a structural issue as gender norms and roles are socially constructed and culturally defined. Health systems barriers identified include difficulty navigating the clinical care system and the need for service integration, particularly with mental healthcare.