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The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo.
Citation: Major T, Menon J, Auyeung G, Soldner F, Hockemeyer D, Jaenisch R, et al. (2011) Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursors. PLoS ONE 6(9): e24687. https://doi.org/10.1371/journal.pone.0024687
Editor: Debra L. Silver, Duke University Medical Center, United States of America
Received: June 15, 2011; Accepted: August 17, 2011; Published: September 22, 2011
Copyright: © 2011 Major et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: VT received funding from National Institutes of Health/National Institute of Neurological Disorders and Stroke (NINDS) and NYSTEM; TM received support from the Starr Foundation (Tri-Institutional Starr Stem Cell Scholars Fellowship). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The derivation of human iPS cells [1], [2] is a major advance for biomedical research including cell-based therapies. However there have been several concerns about the suitability of iPS cells for in vivo applications. Those include a risk of insertional mutagenesis or point mutations in protein-coding regions [3] following the use of integrating vectors, aberrations in epigenetic reconfiguration [4] or incomplete silencing of transgenes with reactivation of potentially oncogenic factors such as c-myc [5]. In addition, questions pertaining to the differentiation potential and in vivo behavior of iPS cells, compared to human ES cells remain unresolved. While iPS cells closely mimic the properties of ES cells, recent studies in the mouse have shown differences in epigenetic state [6], [7] related to cell of origin. Other investigations using the reprogrammable mouse system showed differences in the expression of specific imprinted genes [8] that were correlated with the ability of the cells to contribute to chimeric mice in vivo. A number of studies also showed differences in gene expression between iPS and ES cells, though some of those alterations might be due to residual transgene expression in the resulting iPS cells [9]. A recently developed doxycycline-inducible lentiviral vector system enables transgene excision by Cre-recombinase resulting in transgene-free iPS lines [9]. The lines and specific clones used here have been published and fully characterized by Soldner et al. [9], including an assessment of their pluripotency via factor expression and teratoma formation; their karyotype was also shown to be normal. Interestingly, gene expression studies demonstrated significant differences between excised versus non-excised iPS cell lines with the excised clones more closely matching the gene expression profiles of ESC lines[9]. In this work, we focus on the in vivo behavior of iPS progeny, taking advantage of the availability of iPS lines that are derived from the same fibroblast source and that differ by the ability of actively excising the reprogramming transgenes.
We subjected iPS clones derived, via several reprogramming strategies, from a single fibroblast line (+/− c-myc, excised or non-excised transgenes), to neural induction and differentiation along neuronal, astrocytic and oligodendroglial lineages. Comparisons were established side-by-side with a well-characterized ES line (H9, WA-09). We also asked whether the persistence of viral factor expression could impact more complex interactions of human iPS progeny with the host brain microenvironment in vivo. Using a previously established transplantation paradigm [10] we compared the potential of human neural precursors derived from excised versus non-excised iPS or from control ES cell lines for in vivo integration into the adult rat SVZ and the ability to contribute human cells to the rostral migratory stream and olfactory bulb. Of particular interest was the question of whether residual expression of c-myc might induce excessive proliferation or even promote neoplastic transformation in vivo. Using viral transgene factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines, we observed a comparable differentiation potential along the 3 major neural lineages in vitro, including the derivation of oligodendrocyte and astrocyte lineages. Similarly, the in vivo study demonstrated that neural precursors are capable of responding to local environmental cues by integration into the adult brain subventricular zone (SVZ) independently of transgene excision. These data suggest that carefully validated iPS cell clones are suitable for in vitro and in vivo iPS cell differentiation and for transplantation studies independent of transgene excision.
Neural induction in iPS cells
Induced pluripotent stem cell (hiPSC) lines were generated from a single source of dermal fibroblasts (AG20442) as described previously[9]. In brief, the lines used in the current study include PDB4F-2 and PDB3F-5 which were derived using doxycycline (DOX)-inducible lentiviruses carrying OCT4, SOX2, KLF4 with (4F) or without c-myc (3F). An additional pair of hiPS lines represents a parental line reprogrammed using 3 non-excised factors (no c-myc; PDB2lox-21) and its derivative, subjected to Cre-recombinase mediated excision of reprogramming factors (PDB1lox-21-12). Absence of residual factors in the excised lines was confirmed by Southern blot. The human ES line, H9 (WA-09), was used as a control.
Immunocytochemical analysis of the pluripotency markers showed uniform expression of Nanog, Oct4, Tra1-60 as well SSEA-4 in all hiPSC and H9 lines (Fig. S1A). Neural induction and rosette formation was performed under serum-free conditions by coculture on a stromal cell line (MS5) [11] in the presence of Noggin [12]. Rosettes were harvested mechanically on day 10 of differentiation and replated on culture dishes precoated with polyornithine/laminin in N2 media supplemented with growth factors (P1 rosette stage) (Fig. 1A). Mechanical isolation of rosettes was repeated after seven days (P2 rosette stage). This process selects for clusters of CNS (Central Nervous System) progenitors and greatly depletes the cultures of neural crest precursors. A second selection is performed 15 days later and neural precursor cells (NPCs) are subsequently cultured and passaged for an additional 3 weeks. At day 50 of differentiation, immunocytochemical analysis reveals that >90% of cells were immunoreactive to nestin and Pax6 (Fig. S1B) in all four iPS lines tested as well as the hES control. Immunoreactivity to Ki-67 (44% ±1.7) demonstrated a high proliferative capacity among young neural precursors, comparable across the five lines used (p>0.5). These data suggest that neural induction and neural precursor cell proliferation are not impacted by excision of reprogramming factors or the absence of c-myc.
(a) Schematic representation of the neural differentiation protocol. Phase contrast images of at different stages of neural induction and expansion. (b) Immunofluorescence labeling of neurons (TuJ1/NeuN), astrocytes (GFAP) and oligodendrocytes (O4) following differentiation. (c) Quantification of neurons, astrocytes, and oligodendrocytes derived from human iPS and ES lines. Scale bars 100 µm in a and 20 µm in b. Errors are SD; n = 3 experiments.
Differentiation potential along the three neural lineages
Previous studies have assessed the potential of human ES cell and iPS cells to undergo neural and neuronal differentiation in vitro [13] and the differentiation towards neurons expressing dopaminergic markers [14]. Here we extended those studies to address whether neuronal as well as glial lineages can be derived at comparable efficiencies from excised and non-excised iPS cells and from human ES cells. Neuronal differentiation was induced by withdrawal of EGF/FGF2 and exposure to brain-derived neurotrophic factor (BDNF) and ascorbic acid (AA) for three weeks which resulted in a high yield (>90%) of NeuN+/Tuj1+ neurons. Oligodendrocyte differentiation, as assessed by the expression of surface antigen O4 and appropriate morphology, was induced in the presence of BDNF, AA, PDGFR-AA, sonic hedgehog, FGF-8 and cAMP for six weeks following EGF/FGF2 withdrawal. Quantification of glial phenotypes showed that hES as well hiPS derived NPCs were similarly competent and efficient (56.5% ±1.9) in acquiring oligodendrocyte identity (Fig. 1B, C). For directed differentiation towards an astrocytic fate, NPCs from each of the lines were differentiated in medium containing 5% FBS, for six additional weeks. Again, equal astrocytic potential across all five tested lines was observed by immunocytochemistry for glial fibrillary acidic protein (GFAP, 91.7% ±1.46) (Fig. 1B, C). Thus, neither the number of reprogramming factors used nor their excision impacted in vitro differentiation potential. Furthermore, these hiPS lines exhibited a similar temporal course and capacity for neuro-epithelial, neuronal and glial cell specification, in comparison to human ES cells.
Tranpslantation in the subventricular zone
We subsequently performed in vivo experiments in order to further define a potential impact of reprogramming strategy on in vivo function. We were particularly interested in exploring the in vivo differentiation behavior of iPS and hES cell derived neural precursors. While previous studies have assessed the in vivo survival of pre-differentiated dopamine neuron precursors, here we analyze the site- specific in vivo integration and differentiation potential of naïve neural precursor cells. The response to in vivo environmental cues may be a more sensitive parameter to address the impact of transgene excision and to compare the behavior of iPS and ES cell progeny.
An identical number of NPCs (day 50 in vitro) from each of the hiPS and control lines was stereotactically transplanted into the striatum of adult rats. The animals (n = 20) received a 3-day course of systemic BrdU two weeks post grafting, and were sacrificed at 12 weeks. All surviving animals exhibited a live graft consisting of a core and a large number of migrating cells. Graft volumes as determined by stereological analysis (StereoInvestigator, MBF Bioscience, VT) ranged from 0.95 to 2.8 mm3 (Fig. 2A). The total cell number within the grafts averaged 1.5 ×106 cells (Optical fractionator, range 0.95–2.5×106 cells) (Fig. 2B). One way ANOVA analysis did not demonstrate a statistically significant difference among the volumes and cell counts of the five different groups (p>0.5).
(a) Graphic representation of the volumes of hiPS- and hES-derived grafts. (b) Cell numbers in the core grafts. (c) Migratory behavior of transplanted neural precursors (labeled for human nuclear antigen, hNA, in red). Core grafts from all lines are in the striatum while the majority of cells traveled along the rostral migratory stream (RMS) reaching the olfactory bulb (OB). Scale bars, 100 µm. Errors are SD; total animal number = 20.
The migration pattern of individual cells was captured by NeuroLucida drawings. Human cells were identified using human nuclear antigen (hNA) or human neural cellular adhesion marker (NCAM) expression. They were distributed ipsi-and contra-laterally in the corpus callosum, but the majority of the cells injected traveled forward along the rostral migratory stream into the olfactory bulb, suggesting a response to local subventricular zone cues (Fig. 2C) [15]. There were no differences in migratory patterns among the lines used. To further elucidate the non-random nature of this migratory behavior, we established oligodendrocyte precursor cultures from the H9 line (OPCs, as described above) that were subjected to a FACS sort for O4. O4+ cells were further characterized by the expression of olig2, thus confirming their oligodendroglial identity (Fig. S2). The sorted cells were injected at the same coordinates and the animals sacrificed at 12 weeks (n = 6). The migration routes exhibited by the O4 cells were very distinct from those of the naïve neural precursor cells and specifically followed the white matter pathways along the corpus callosum and the internal capsule. In contrast to the neural precursor cell grafts there were only very few cells traveling along the RMS. The cell migration paths are illustrated in representative camera lucida drawings (Fig. 3A).
(a) Representative camera lucida drawings of core grafts and migrating cells, highlighting that OPC grafts were confined to the white matter tracts while NPCs migrated to the RMS and the OB. (b) Within the graft cores, cells were predominantly neuronal expressing doublecortin (Dcx; PDB4F-2) and TuJ1(PDB3F-5). Migrating cells in the corpus callosum were immunopositive for CD24 (PDB2lox-21) and a smaller proportion labeled for NG2. In the SVZ, grafted cells acquired region-specific phenotypes: type A cells (EGFR/CD24; PDB1lox-21-12), type B cells (EGFR/GFAP; PDB3F-5) as well transit amplifying type C cells (EGFR/Dlx2; PDB2lox-21). Within the olfactory bulb (OB) rare human cells exhibited an interneuron phenotype, expressing calretinin (PDB3F-5) or GABA (PDB4F-2). Scale bars, 20 µm. hNCAM = human-specific neural cell adhesion molecule; hNA = human nuclear antigen.
In vivo phenotypes
The core grafts in all the experimental and control groups were mainly located in the striatum, with small clusters occasionally seen around the corpus callosum. Most of the cells in the core expressed the human markers, human nuclear antigen or human NCAM, were neuronal precursors expressing neuron-specific class III beta-tubulin (Tuj1) as well doublecortin (Dcx)+,(>60%) (Fig. 3B, upper panel). A smaller population of glial cells recognized by expression of the NG2 chondroitin sulfate proteoglycan was also observed. GFAP+ human cells were rare and mostly located in the periphery of the core graft (<1%). Host GFAP+ cells were found along the injection site. Human cells that were located away from the core acquired phenotypes typical of the local environment. Human iPS- as well control-derived neural precursors exhibited region specific immunohistochemical profiles, and integrated along the SVZ, giving rise to the three SVZ niche phenotypes [16]. A small proportion of human cells were identified within the ipsilateral and contralateral SVZ where they expressed markers characteristic of the activated type B stem cell population (EGFR+/GFAP+) or type C transit amplifying cells (EGFR+/Dlx2+) [17] (Fig. 3B, middle panel). A majority of cells however exhibited a type A or migrating neuroblast phenotype, labeling for CD24, PSA-NCAM and Dcx (Fig. 3B, middle panel) as they travelled anteriorly along the RMS. Human cells could be seen at the distal RMS, entering the olfactory bulb, whereby a few cells (<1%) migrated to the granular and periglomerular layers. These cells differentiated into calretenin or calbindin periglomerular interneurons, or γ-aminobutyric acid (GABA) expressing granular interneurons (Fig. 3B, lower panel). The number of cells exhibiting interneuron phenotypes was very small (one or two per section) but identical among all hiPS and hES lines used and similar to previously reported data with human ES cells [10] and primary fetal neural progenitors [18]. This region-specific immunohistochemical profile of human cells is identical to that exhibited by endogenous SVZ stem cell progeny and was demonstrated in all 4 hiPS clones and hES line used, as shown in Figure 2 (cell of origin is indicated in the legend). All co-localisations were confirmed by serial 3D reconstruction of confocal sections. No human cells were found in other regions of the olfactory lobe, suggesting that they reached the olfactory bulb selectively by the RMS route rather than by random migration. There was also an absence of neuronal differentiation outside the core graft and the RMS/olfactory area. In contrast the transplanted oligodendrocyte precursors formed a smaller core and exhibited migration along the corpus callosum without any evidence of integration in the SVZ or expression of markers suggestive of SVZ stem cell progeny (Figure S2). The OPCs did not reach the olfactory bulb in any of the animals injected.
The immunohistochemical profile of the neural precursors prior to transplantation demonstrates immunoreactivity to nestin and TuJ but not EGFR, Dlx2, and CD24 (Figure S3). Similarly the oligodendrocyte progenitors were immunopositive for O4 and olig2 but did not exhibit any SVZ markers. These data suggest a significant cell-autonomous response exhibited specifically by cells at the neural stage in response to environmental cues.
Proliferation in vivo
The BrdU regimen involved pulse labeling ten weeks prior to sacrifice followed by a chase period for the remainder of the time, allowing identification of label retaining cells and neurons born in vivo. Concomitant labeling with Ki67 allows us to determine the actual rate of proliferation in vivo following graft integration. All five lines tested exhibited BrdU retention and a similar percentage of BrdU+ cells in the graft core (average 19.9% ±6.6; range: 17–23%; calculated stereologically via optical fractionator)[19] (Fig. 4A). The predominant phenotype of the BrdU positive cells in the core graft was neuronal (Fig. 4B). In the SVZ, the highly proliferative transit amplifying C cells were BrdU negative indicating label dilution. A few human cells residing in the SVZ retained BrdU and co-expressed EGFR, compatible with the slowly dividing type B stem cells. The BrdU label was also retained by the neuroblasts traveling to the RMS (Fig. 4B). All grafts from the experimental and control groups exhibited a proliferation capacity of less than 1% as shown by Ki-67 staining (Fig. 4A), indicating that most cells stopped dividing 12 weeks post grafting, despite an initial period of proliferation as indicated by the BrdU labels. None of the grafted animal brains harbored a teratoma (absence of alpha-fetoprotein, cytokeratin, myosin) or other type of tumor as assessed by histological analysis on hematoxylin and eosin (H&E) sections.
(a) Quantification of BrdU and Ki67 positive cells in the core grafts from hiPS and hES lines. Representative immunohistochemical labeling for BrdU (PDB3F-5) and Ki67 (PDB4F-2). (b) BrdU label retention and Ki67 within the graft core, SVZ and RMS. In the core graft BrdU label was mainly found in the neuronal cells (BrdU/NeuN; PDB2lox-21). In the SVZ BrdU was mostly retained by the type B cells (BrdU/EGFR; PDB4F-2) while in the RMS some of the migrating neuroblasts (BrdU/CD24-PDB3F-5) are BrdU positive. Ki67 labeling was very low in all regions. Scale bars, 20 µm. Error bars are SD; n = 20.
Reprogramming strategies have evolved rapidly, including the use of micro-RNA [20], non-integrating vectors [21]–[24], mRNA [25], protein [26], [27] or small molecules [28] in lieu of viral transgenes, or microenvironmental modifications to induce endogenous genes [29]. However reprogramming efficiency via most of these methods is extremely low, in the range of 0.001%. The use of integrating vectors offers a relatively more efficient means of reprogramming but also raises safety concerns with the subsequent risk of genetic and epigenetic aberrations [3], [4], the overexpression of proliferation- and potentially neoplasm-associated genes such as c-myc[30] and the incomplete silencing of reprogramming factors following differentiation[5]. Such issues would constitute barriers to the clinical use of iPS progeny [31]. In fact, transmission of iPS clones through the germ line in mice has resulted in tumor formation in 20% of the chimeric progeny, probably due to reactivation of the c-myc transgene [32]. The use of doxycycline-inducible vectors allows greater control over transgene expression but does not eliminate the possibility of residual transgene expression, due to leaky promoters. Thus several groups have introduced integrating vectors that incorporate loxP sites allowing their excision via Cre recombinase once pluripotency is achieved [33]–[35] [9]. Such systems provide the advantages of integrating vectors and higher reprogramming efficiency while allowing for definitive elimination of full transgene expression. Short sequences of vector DNA however remain integrated in the host genome with the potential to interfere with normal function [36].
Despite the rapid evolution of reprogramming strategies, their impact on the potential of iPS cells to stably differentiate along specific lineages has not been extensively studied. Recent literature has reported variable "innate" propensity of ES and iPS lines for preferential differentiation along different lineages [37], as well as interline differences in efficiency of neural induction [13]. Other investigators have suggested that factor-based reprogramming can leave an epigenetic memory of the tissue of origin that may impact directed differentiation [6]. Our data however demonstrate robust neural induction and differentiation into neural and glial lineages, comparable in human ES and iPS lines. This suggests that "innate" differentiation propensity may be related, at least in part, to variations in the specific culture protocols used. A more recent report suggests a similar range of capacity towards motor neuron differentiation [38] in a set of iPS lines derived from 7 different sources, and yet another publication identifies a role for miR-371-3 in specifying the propensity towards neural differentiation [39].
Differentiation along glial lineages is an important tool in disease modeling particularly when non-cell autonomous factors are significant contributors to disease phenotype as is the case in ALS [40], [41], or in cell-based approaches to remyelination. High yield derivation of astrocytes or oligodendrocytes has not been reported to date from human iPS cells. Here, we demonstrate the generation of high percentages of astrocytes (>90%) and oligodendrocytes (>50%) in vitro from all ES and iPS clones presented. The protocols used are based on previously published methods [11], and emphasize the selection of CNS precursors at the expense of neural crest progeny. This is a crucial step in ensuring a higher yield of glial progeny since neural crest derivatives are highly proliferative competitors in culture. However this rate-limiting step relies on mechanical selection based on morphological criteria, and is thus operator-dependent. Ongoing efforts aim to optimize this component of the differentiation protocols, by substituting small molecules or marker-based selection of the cells of interest. Once the cultures are enriched in neural precursors, astrocyte differentiation is achieved via mitogen withdrawal and the addition of serum over several weeks. Oligodendrocyte production is highly dependent upon the elimination of exogenous bFGF and maintaining high concentrations of sonic hedgehog and FGF-8. These protocols exhibit a high reproducibility rate and have in fact led to comparable results among excised and non-excised clones. Remarkably all iPS clones exhibited a profile highly similar to that seen in H9, a well-characterized ES line with a significant neural propensity. Thus reprogramming strategy such as excision of viral transgenes, inclusion of c-myc did not impact in vitro differentiation of human iPS cells along neurons and glia. Obviously this study does not compare multiple lines but is focused specifically on the role of factor excision in lines that exhibit the same background.
The in vivo behavior of iPS cells has not been studied extensively to date. Here we grafted hES and hiPS derived neural progenitors in the adult brain and analyzed their ability to interact with a complex in vivo microenvironment. While the factors that control the cells' response to cues emanating from the SVZ are not well defined, previous experiments using human fetal neural precursors or hES progeny have demonstrated a remarkable ability for a fraction of cells to integrate that niche. Cells have acquired a neuroblast or a transient amplifying cell phenotype and, very infrequently, a stem cell phenotype that retained BrdU label up to 10 weeks. The human iPS-derived neural precursors also followed the SVZ-olfactory bulb migratory pathway, giving rise to more differentiated neurons and reaching the olfactory glomeruli. Olfactory neurogenesis in the rat is an inefficient phenomenon, with large numbers of cells generated in the SVZ but a very rare percentage incorporating the olfactory glomeruli and differentiating into interneurons. In comparison, human iPS-derived oligodendrocyte precursors followed the white matter along the corpus callosum and did not engage along the RMS or reach the olfactory bulb, suggesting that response to the SVZ niche is in part cell-autonomous. Importantly, the SVZ niche phenotypes exhibited by the iPS derived NPCs were acquired in vivo, likely in response to the local environment.
Other important data points include the absence of excessive proliferation regardless of c-myc status. As expected, NPCs exhibit significant proliferation shortly after transplantation as shown by BrdU retention within the graft core. When examined 12 weeks after grafting, however, proliferation had decreased dramatically as demonstrated by the rare Ki67 expression. More importantly there were no differences in survival, SVZ integration and migration along the RMS and neuronal differentiation among the hiPS lines, all 4 of which behaved similarly to the human ES line. The lines used in this study have also been differentiated into dopamine neurons by Jaenisch and colleagues and transplanted in rat models of Parkinson's disease with modest survival and some behavioral amelioration [14].
Our data suggest low probability of transgene reactivation or neoplastic transformation via the use of doxycycline-inducible lentiviral vectors to deliver reprogramming transgenes. The inclusion of c-myc in reprogramming did not result in altered proliferative behavior in vitro or in vivo. In addition, Cre-mediated excision did not have a definitive impact on differentiation potential or in vivo behavior of iPS neural progeny. However, more sensitive in vivo paradigms and outcome parameters or longer term studies may be necessary to detect more subtle differences. It is also conceivable that higher levels of transgene expression as in non-inducible vectors may highlight subtle differences among cell lines.
Reprogramming strategies, such as the specific transgenes used, their copy number or their excision had no impact on the ability of hiPS cell progeny to respond to in vivo cues and to acquire regional phenotypes within the adult brain. Neither the use of c-myc in reprogramming nor its excision impacted the proliferation rate or graft size of human iPS progeny in vivo. This result cannot rule out that stronger transgene expression would affect in vivo behavior as might be the case with other delivery systems or non-inducible vectors. The use of a single fibroblast source does allow for a clear comparison of the impact of transgenes but future studies will need to address this issue across additional lines.
All animal experiments were done in accordance with protocols approved by our Institutional Animal Care and Use Committee (IACUC) and following the National Institutes of Health (NIH) guidelines for animal welfare. The study was also approved by our institutional Ethics committee (ESCRO) under Protocol 2009-06-005.
Cell culture
Undifferentiated hES (H9 (NIH code: WA09; Wisconsin Alumni Research Foundation, Madison, WI) and hiPS cells (PDB4F-2, PDB3F-5, PDB2lox-21, PDB1lox-21-12) were cultured under growth conditions as described previously [42] on mitotically inactivated mouse embryonic fibroblasts (MEFs; GlobalStem, Rockland, MD). The iPS lines were derived from fibroblasts obtained from the Coriell Repository (AG20442; Coriell Institute for Medical Research, Camden, NJ) by Soldner et al [9]. The derivation of the lines as well as the lentiviral vector design and cre-mediated excision are described in detail in Soldner et al. [9]. For neural induction, hES cells were plated on a confluent layer of irradiated stromal cells (MS-5) in knock-out serum replacement (KSR, Invitrogen) medium supplied with Noggin (250 ng/ml). Medium was changed every two days until day 8, when Noggin was replaced with mouse supersonic hedgehog (msSHH, 20 ng/ml) and Fibroblast growth factor (FGF8, 100 ng/ml). All growth factors were purchased from R&D Systems, Minneapolis, MN. Rosette structures were harvested mechanically at day 10 of differentiation (termed Passage 0; P0) and gently replated on 15 µg/ml polyornithine/1 µg/ml Laminin (Po/Lam) coated culture dishes in N2 medium (termed Passage 1; P1). Medium was changed every 2 days, and was supplemented with growth factors as follows: 20 ng/ml msSHH, 100 ng/ml FGF8, 20 ng/ml brain-derived neurotrophic factor (BDNF) (all R&D Systems), and 0.2 mM ascorbic acid (AA)[11] (Sigma–Aldrich, St Louis, MO). After 7 days of P1 culture, rosettes were re-picked and re-plated on Po/Lam coated dishes and kept for an additional week in N2 media containing BDNF, AA and msSHH (termed Passage 2; P2). At the end of the P2 stage, cells were exposed to Ca2/Mg2-free Hanks balanced salt solution (CMF-HBSS) for 20 min at 25°C and spun at 200 g for 5 min. The cell pellet was gently resuspended in N2 media and plated on PO/lam coated dishes (termed Passage 3, P3). Starting with P3, neural precursor cells (NPCs) were maintained in N2 medium supplemented with 20 ng/ml of fibroblast growth factor 2 (FGF2) and 20 ng/ml of epidermal growth factor (EGF) that was changed every 2 days. As of this stage, the cells will loose rosette formations and form clusters of neural progenitors interspersed among flat cells. The cultures are subsequently passaged at least twice by manual picking of the clusters of central nervous system (CNS) precursor cells. This mechanical purification step ensures the generation of cells highly enriched in CNS precursors and largely devoid of neural crest lineages. Transplanted cells are harvested at this stage.
For neuronal differentiation hES cell-derived CNS NPCs, were subjected to EGF/FGF2 withdrawal at day 50, and cultured for an additional three weeks in N2 media containing BDNF(20 ng/ml) and AA (0.2 mM). For astrocyte differentiation, hES cell-derived NPCs were kept six weeks in N2 media supplemented with 5% Fetal Bovine Serum (FBS, Invitrogen), after mitogen withdrawal. For oligodendrocyte differentiation, after EGF/FGF2 removal, hES cell-derived NPCs were cultured for an additional six weeks in N2 media that contained BDNF (20 ng/ml), AA (0.2 mM), msSHH (100 ng/ml), Fibroblast growth factor (FGF8, 100 ng/ml), Platelet-derived growth factor-AA (PDGF-AA, 20 ng/ml) and dibutyrilic 3′-5′-cyclic adenosine monophosphate, (dbcAMP, 0.2 µM).
For the transplantation of the oligodendrocyte precursor cell (OPCs) population, 100- day old NPCs were dissociated with Accutase (Innovative Cell Technologies, San Diego, CA) and subjected to FACS using O4 antibody (1∶200; Millipore, Billerica, MA) on a MoFlo flow cytometer (Cytomation, Fort Collins, CO).
Animal surgery
Young adult Sprague Dawley female rats (3 months old at time of grafting, n = 26) were acquired from Taconic and used throughout the study. Stereotactic intracranial transplantation of graft cells was performed under full anesthesia using a mixture of ketamine and xylazine (Hospira, Lake Forest, Illinois/Inc/Ben Venue Laboratories). For all the implants, the animals received a unilateral 1 µl (250,000 cells in sterile HBSS) injection at the following coordinates: striatum: anteroposterior, + 1.6; mediolateral, -1.5; ventral, - 4.2; tooth bar, -2.3. All coordinates relative to bregma and ventral coordinates relative to cortex.
All rats received cyclosporine (Neoral 100 mg/ml; Novartis, Basel, Switzerland) at 20 mg/kg/day intraperitoneally. This regimen was initiated 1 day before grafting and maintained until the animals were sacrificed, 12 weeks following transplantation.
BrdU administration
The BrdU (Sigma-Aldrich) regimen consisted of two doses of BrdU at 150 mg/kg administered every 12 h for a total of 2 doses over 3 days. This regimen was initiated 2 weeks after transplantation in the SVZ. The animals (n = 26) were sacrificed 10 weeks after the administration of BrdU. BrdU was dissolved in sterile normal saline and .007 M NaOH.
Tissue processing
Rats were deeply anesthetized with a 25-mg intraperitoneal injection of pentobarbital solution (Nembutal Sodium Solution, Abbott Laboratories Abbott Park, IL). This was followed by transcardial perfusion with 10% Sucrose saline at 4°C (Aldrich-Sigma) followed by 4% paraformaldehyde (PFA) in PBS also at 4°C (pH 7.4). The brains were carefully extracted, post-fixed overnight in 4% PFA at 4°C, and subsequently transferred to 30% sucrose at 4°C until embedding. Optimal cutting temperature compound (O.C.T. Compound, Tissue-Tek, Sakura Finetek, Torrance, CA) was used to embed the brains and sections were cut on a freezing cryostat and stored at −80°C.
Sections were washed briefly with PBS/0.1% BSA (Sigma-Aldrich). For fluorescence double immunohistochemistry, sections were first blocked with 10% Normal Goat Serum (Invitrogen, Carlsbad, CA) in PBS and 0.3% Triton X-100 (with the exception of surface antigens, where Triton X-100 was omitted). Some antibodies required a pretreatment step as follows: 30 min in 2N HCl at 25°C for Brdu; 3 min in 100% acetone at −20°C for human nuclear antigen. Primary antibodies were incubated overnight at 4°C. Appropriate secondary antibodies and fluorochromes (Alexa conjugates (Molecular Probes, Invitrogen) were applied for 1 h at 25°C followed by PBS washes, DAPI (Molecular Probes) counterstain and mounted in glycerol. The primary antibodies included: rat anti-Brdu (1∶40, Abcam, Cambridge, UK); Calretinin(1∶2000, Swant, Bellinzona, Switzerland); Calbindin(1∶1000, Swant); EGFR(1∶100; Santa Cruz Biotechnology, Santa Cruz, CA),PSA-NCAM (1∶800, Millipore); Nestin (1∶200 Millipore); CD24 (1∶100, BD Pharmingen, San Diego, CA); Dlx2 (1∶200 Abcam, Cambridge, CA); Tuj-1(Covance 1∶200, Princeton, NJ); human NCAM (Eric-1, 1∶100, Santa Cruz Biotechnology); and human nuclear antigen (1∶50 Chemicon), GFAP(1∶5,000; Dako, Carpineria, CA), NG2 (1∶100; Chemicon), DCX (1∶500 Abcam), O4 (1∶200 Millipore).
Graft volumes were estimated using the Cavalieri estimator probe (StereoInvestigator-6, MBF Bioscience, Williston, VT). Total cell number was assessed in the graft core. Systematic random sampling was applied to the regions of interest (graft core and areas of distribution of human cells) as defined on serial sections. The stereological software was used to design and implement the fractionator probes at a coefficient of error (Gunderson) of ≤0.1.
Supporting Information
Figure S1.
(a) Immunocytochemical characterization of hiPS cell lines and H9 line for the expression of the pluripotency markers Tra1-60, Nanog, Oct4 and SSEA-4. (b) Immunofluorescence staining of neural precursors derived from four different hiPS clones and H9 line. Neural precursor cells (NPCs) at 50 days in vitro show high expression of Nestin and Pax6. Scale bars, 20μm.
Figure S2.
hES or hiPS derived oligodendrocyte precursor cells used for grafting were immunolabeled for the oligodendrocyte cell surface marker O4 (hNCAM/O4) while the markers specific for SVZ type A cells (hNCAM/Dlx2), type B (hNCAM/hEGFR) or type C cells (hNA/Dcx), were absent. O4 cells co-labeled for Olig2. Nuclear counterstain (DAPI) in blue. Scale bar, 20μm.
Figure S3.
In vitro immunocytochemistry of hES and hiPS derived neural precursors for phenotypic markers of the SVZ. NPCs (hiPS4) 50 days old were mostly immunoreactive for Nestin and Tuj1 with a low percentage of doublecortin (Dcx) expressing cells. At this stage, neural precursors were negative for markers specific for SVZ type A cells (Dlx2/CD24), type B (hEGFR/GFAP) or SVZ type C cells (hEGFR/Dlx2). Scale bar, 20μm.
We would like to thank Dr Mark Tomishima, director of the Stem Cell Core Facility at the Center for Stem Cell Biology at Memorial Sloan Kettering Cancer Center for technical assistance and advice.
Author Contributions
Conceived and designed the experiments: VT RJ. Performed the experiments: TM JM GA FS DH. Analyzed the data: TM VT JM. Contributed reagents/materials/analysis tools: DH FS RJ. Wrote the paper: TM JM VT.
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Pardee UNC Health Care operates urgent care facilities, conveniently located close to the communities we serve. These locations are open every day, including holidays and accept most major insurances. Plus, if you require follow-up care and choose a Pardee provider, your medical records, including lab reports, X-ray images and prescriptions written, will be readily available through My UNC Chart.
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Late one night time in early 2000, the members of Blonde Redhead had been in a barn exterior of Seattle that had been transformed right into a recording studio. The New York–primarily based indie rock trio had been nearly completed making their fifth album, Melody of Certain Damaged Lemons. They had already completed a melancholy tune known as "For the Damaged" the place co-lead vocalist Kazu Makino sang resignedly over delicate piano and a sparse acoustic guitar. They'd additionally recorded a extra dramatic, prolonged piano vamp for the tune, however weren't positive what to do with it. Then both Makino or her bandmate Amedeo Pace — they will't keep in mind which — had the concept so as to add wordless vocals of the tune's melody: aaaaah-aah-aah-aah-aaah-aah-aah. Makino initially envisioned it being completed by a choir with a number of kids, however they couldn't afford that. Instead they layered about 20 takes of Makino doing it herself, changing into, as she says, "a military of my voice."
The mission had been delayed, and earlier within the recording course of, their label Touch & Go informed them they wanted to get the album artwork completed. Makino figured they'd in all probability find yourself with ten songs, so she wrote out a convoluted run-on sentence and chopped it into ten elements for the monitor titles. This new tune would turn out to be the album's eleventh monitor, so it went unlisted on the again of the packaging. Just over two-and-a-half minutes lengthy, it was named "For the Damaged Coda." Thinking again to that late night time within the studio, Makino says, "Because you're in such a cocoon, you've gotten this very robust sensation that nobody goes to hearken to this music. Once that's completed, you understand, 'Wow, that is really going to go on the market.' And past that, mainly it's out of your fingers."
Fourteen years later, "For the Damaged Coda" was used within the ultimate moments of an episode of Adult Swim's Rick and Morty in the course of the cartoon's first season. Makino says she solely vaguely remembers being approached to approve it. The tune soundtracked the revelation that (okay, bear with me) an Evil Morty was the mastermind behind a plot to journey throughout alternate dimensions, killing Ricks and kidnapping different Mortys to get the eye of the present's central Rick, and now that Evil Morty was roaming undetected within the Citadel of Ricks, an intergalactic society ruled by the Council of Ricks. Then throughout final yr's third season, after Rick and Morty had amassed a now fanatical fan base, "For the Damaged Coda" performed as soon as once more because it turned clear that Evil Morty had engineered a conspiracy to get himself elected because the chief of the Citadel of Ricks and homicide anybody who may problem his energy.
In the next weeks, "For the Damaged Coda" was streamed and downloaded so many instances that it reached No. 15 on Billboard's Hot Rock Songs chart, 17 years after it was first launched. Over the course of their profession, the music of Blonde Redhead has been utilized in motion pictures, commercials, and TV reveals, even getting a coveted placement on Grey's Anatomy again in 2007, however none of those syncs had a cultural impression wherever close to what occurred with Rick and Morty.
Today, "For the Damaged Coda" has been streamed over 27 million instances on Spotify, which is 23 million extra instances than the second-most streamed Blonde Redhead tune. For some extra perspective, probably the most streamed tune on Spotify by Sonic Youth, the indie rock pioneers and the band that Blonde Redhead was most in comparison with in 2000, is at near 18 million. The most streamed tune by punk godheads Fugazi, whose member Guy Picciotto co-produced Melody of Certain Damaged Lemons, is at simply over 16 million.
"For the Damaged Coda" additionally turned a meme, enjoying over clips of Fortnite fails and dopey characters getting clowned on SpongeBob SquarePants. Makino says a buddy's 12-year-old daughter in Italy informed her that youngsters in her class sing the wordless melody at one another when somebody makes a mistake, and so they don't even watch Rick and Morty. "It actually makes you are feeling like you haven't any say in what occurs in your profession," says Blonde Redhead's drummer, Simone Pace. "The remainder of your profession you simply battle to make good information, go on tour, then one factor like this has extra impression than something."
Across its three seasons. Rick and Morty's co-creator Justin Roiland (Dan Harmon is the opposite) has additionally chosen surprising tracks by bands like Belly, Mazzy Star, and Chaos Chaos for emotional, episode-ending sequences. In every case, this placement has resulted in an outsize spike in curiosity within the tune. Last week, Sub Pop Records launched a soundtrack for Rick and Morty that's principally a showcase for its versatile composer, Ryan Elder, however it is going to additionally function every of those choices. Rick and Morty has developed into Adult Swim's hottest present of all time, although there have solely been 31 episodes over nearly 5 years. Its followers have gotten the rep as a few of the most devoted (some may say obnoxiously so) followers in popular culture, although there have been cases when a subsection of them have resorted to vile misogyny. But contemplating that there's now a marketplace for formally licensed Rick and Morty plush slippers and toy dimension portal weapons, the truth that they might simply push a tune's streaming numbers into the thousands and thousands isn't that shocking.
While getting a tune on a movie or TV present has lengthy been a reliable method for musicians to achieve consideration, on this period of diminished gross sales and a glut of different music each new and outdated to compete with, these placements nonetheless stay essential. Phenomenon like "For the Damaged Coda" or MIA's "Paper Planes" within the Pineapple Express trailer have turn out to be increasingly more uncommon, however nearly any mainstream publicity or free promotion helps.
Belly's frontperson Tanya Donnelly says that again within the '90s, the band's profession was bolstered by soundtrack appearances in movies together with Mallrats, Twister, and Tank Girl. Their cowl of Tom Jones's "It's Not Unusual" was used within the Brendan Fraser car With Honors, which is mainly a triple-word rating for the period. But getting a tune on Rick and Morty in 2014 arguably was extra essential for them.
Belly was considered one of Justin Roiland's favourite bands when he was an adolescent, so he selected their love tune "Seal My Fate" for when Rick and Morty got here closest to approximating a giant, rom-com second inside its notoriously darkish universe. After it was launched in 1995, "Seal My Fate" turned the last-ditch fourth single from King, Belly's commercially unsuccessful follow-up to their breakthrough debut. Now it's the band's second-most streamed tune on Spotify after the Buzz Bin staple, "Feed the Tree."
Belly acquired again collectively in 2016 after a 20-year absence, and Donnelly says that they've met some children throughout their post-reunion gigs who admit they realized concerning the group by means of Rick and Morty. Once throughout a present in Chicago, she interrogated a younger fan about how he knew about her music and he stated it was as a result of a streaming web site's algorithm really useful her when he was enjoying Sarah McLachlan. "There's much less genre-identification and time-identification," Donnelly says of the listening habits of youthful music listeners. "I really feel like those that use a streaming web site as their predominant supply of discovery aren't fairly as ageist as my era might need been."
While a lot of the songs utilized in Rick and Morty seemingly come from Roiland's outdated CD assortment, the trendy indie synth band Chaos Chaos, comprised of sisters Asya and Chloe Saavedra, has turn out to be frequent collaborators with the present's creators. Roiland was a fan of Smoosh, the group the sisters fashioned earlier than they had been youngsters, and he approached them after they launched Chaos Chaos's second EP, saying he wished to make use of the atmospheric monitor "Do You Feel It?" for a scene within the second season during which Rick makes an attempt suicide and fails. The group didn't know concerning the present and didn't see the e-mail for some time earlier than they responded. "It nearly looks as if it might simply haven't occurred," says Asya.
Now the tune has over 24 million streams on Spotify. The feedback for the Committed to the Crime itemizing on Bandcamp are plagued by Rick and Morty references like "Unity introduced me right here" and "WHAT UP MY GLIP GLOPS."
"Memories," one other Chaos Chaos tune, appeared within the present's second season, and within the third, Roiland requested to make use of some instrumental music the sisters had made for the brand new monitor, "Terryfold," which made it to No. 33 on Billboard's Hot Rock Songs chart. They additionally added vocal harmonies to the Dan Harmon–sung "Fathers and Daughters (Doo-Doo in My Butt)." "I'm okay with not taking credit score for that tune," says Asya.
At the merch desk after reveals, Chaos Chaos say they get lots of people telling them they acquired into them by means of Rick and Morty, and so they have to recollect to incorporate "Do You Feel It?" of their setlists since they know folks in all probability got here to see them play it. The group self-releases their music, in order that they see the all particulars of their streaming numbers and the way a lot cash has are available. Still they had been shocked after they acquired the test after the "Do You Feel It?" episode aired. "We really thought there was an issue. We thought it was a glitch," says Chloe. "We noticed the quantity and we emailed the corporate, 'Okay guys, we predict one thing perhaps went flawed. We acquired this large chunk of cash and we wish to test in to know what's up with that.'"
The members of Blonde Redhead say the royalties from the rise of their streaming numbers haven't amounted to a considerable change within the sum of money they've acquired. They've been constantly touring for greater than 20 years and so they say the age of their followers have all the time been combined, so if it's getting any youthful due to Rick and Morty, they haven't seen. They additionally haven't been capable of e book any greater venues due to the tune's notoriety. In 2017, the group carried out Melody of Certain Damaged Lemons in its entirety at FYF Fest in Los Angeles, however they assume the promoter requested them to take action due to his personal affection for the album fairly than any attainable Rick and Morty tie-in.
Even in the event that they haven't seen any tangible modifications of their profession from the unfold of "For the Damaged Coda," thus far the results have been extra psychological than sensible. "I by no means wished to be labeled area of interest, and even indie rock or experimental or one thing like that," Makino says. "It was such a distant dream of mine that we might write music that appealed to everybody, nevertheless it by no means, ever occurred. But now, after that occurred, I'd get on the subway and I'd assume to myself, 'There are literally individuals who know that tune on this automotive.'"


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An agreement has been signed between the Government of South Africa and the Hungarian Government offering 100 scholarships to South African students to study at participating public university in Hungary. Most of the available courses are taught in English. Students applying for studies conducted in Hungarian will be required to do a one-year preparatory course before commencing with their degree studies.
At degree level, 30 students can apply, 60 for Master and 10 for PHD. The application process is currently open and closes on 5 March 2017.
South African citizens in good health are eligible to apply, with a strong academic record or a minimum average of 60%. Candidates with an interest in studying in Hungary and demonstrate commitment to the development of South Africa and candidates must be available to study in Hungary from September 2017.
All applicants must meet the entry criteria for their selected programme in Hungary and must meet the minimum academic requirement for entry into a similar programme at a South African university.
Fields available for the bachelor degree are sciences and sustainable environment, engineering and information technology, agricultural science and food science.
For the Masters, available is the natural sciences especially biotechnology, bioenergetics and renewable energy, engineering especially mining engineering, information technology; economics, business and management studies and tourism; agricultural science and food science; physical education and coaching, and adult education, and human resources development; international relations and European studies; and Hungarian language studies.
For the PHD all fields of study are accommodated.
Application process for South African Applicants
The application process is two-fold and both applications must be submitted in order to be considered:
- Complete the DHET application form, available to download here and email only the completed and signed form email@example.com.
- Complete their application and upload all supporting documents online on the Tempus Public Foundation website on www.stipendiumhungaricum.hu. (open from end of January).
Candidates applying for programmes taught in Hungarian need to complete a one year language preparatory course and should apply for the language course only, not for the degree. Applications for the degree programme will be done later, if admitted.
PHD applicants are required to visit this website before they submit their application. The website provides information about the Hungarian doctoral schools, available research field and research topics as well as the supervisors of the doctoral students. Thesis topic proposals are also available here.
Documents and links
A list of available courses and institutions is available here
Information about studying and living in Hungary is available here
Guide for the Hungarian online application
List of available courses and institutions
Website to apply for language preparatory course
Information for PhD candidates
Information about studying and living in Hungary


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Breaking news and expert analysis on legal and compliance issues
From Health Law Daily, April 23, 2015
By Kayla R. Bryant, J.D.
Patients admitted to the hospital on the weekends are 25 percent more likely to develop hospital-acquired conditions than those admitted on weekdays, after adjusting for several different factors, according to a report issued by the BMJ. Treatment for this group of 14 conditions, termed "never events," does not qualify for reimbursement from federal health care programs, as the conditions are the result of "serious, costly, and preventable medical errors," according to CMS. However, some in the industry are of the opinion that not all hospital-acquired conditions are preventable and that CMS' policy negatively impacts patient care.
Study. This study involved data from the Nationwide Inpatient Sample assembled by the Agency for Healthcare Research and Quality, the largest inpatient database in the U.S., which is representative of all types of payers and captures 20 percent of all hospital discharges. The study included all patients admitted between 2002 and 2010. Each discharge was considered an independent event, as unique patient identifiers were not available. The frequency of individual hospital-acquired conditions was tabulated and compared to inpatient charges and prolonged stays.
Results. Out of 351 million inpatient admissions during the studied time period, 4.1 percent involved at least one hospital-acquired condition. According to the report, 5.7 percent of these occurrences happened to patients who were weekend admissions, as opposed to 3.7 percent who were admitted during the week. Weekday admissions occurred mostly for routine problems, and weekend admissions largely stemmed from the emergency department. From 2002 to 2010, hospital-acquired conditions increased by 1.6 percent. The presence of one of these conditions was associated with both higher charges and prolonged hospital stays.
Discussion. Although a 17 percent reduction in hospital-acquired conditions has resulted in savings of approximately $12 billion, an estimated $9.3 billion still goes to treating potentially preventable medical errors. The decreased staff volume and level of medical expertise available during the weekends are possible factors contributing to the increase in hospital-acquired conditions among patients admitted on weekends. On weekends, diagnostic testing and operative intervention are allocated fewer resources, and certain procedures are often delayed longer during these days. This trend is not limited only to the U.S., as studies from other countries have shown that the mortality rate in hospitals increases for weekend admissions.
The report indicated that additional resources and attention from health care administration are needed to increase the quality of care provided during the weekends. These conditions are generally attributed to medical errors, so increased training, plans for handling reduced staffing, and strong adherence to safety may help prevent these conditions. An estimated 20 percent of hospital-acquired infections are deemed to be preventable if proper training procedures are implemented.
Reduction program. Hospitals now have more of an incentive to prevent these conditions. Section 3008 of the Patient Protection and Affordable Care Act (ACA) (P.L. 111-148) implemented the Hospital Acquired Condition Reduction Program. Hospitals are scored according to performance. Those hospitals that rank in the quartile with the highest total scores receive reduced payments from CMS beginning in fiscal year (FY) 2015.
Companies: The BMJ
MainStory: TopStory ReimbursementNews CMSNews AuditNews IPPSNews MedicaidPaymentNews PartANews RiskNews
Introducing Wolters Kluwer Health Law Daily a daily reporting service created by attorneys, for attorneys providing same-day coverage of breaking news, court decisions, legislation, and regulatory activity.


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Published at Wednesday, March 20th, 2019 - 16:32:35 PM. Types of car. By Adeline Winkler.
Comprehensive Coverage: This cover the damage you cause to other people either injury damage other people s property in respect to an accident.
Finally there is comprehensive coverage. This kind of insurance covers you if there is some kind of problem when you are not in the car. For instance if a tree falls on your car comprehensive coverage has got your back. If vandals smash in your windshield you have protection.
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Any content, trademark's, or other material that might be found on the Jennifer-thomas website that is not Jennifer-thomas's property remains the copyright of its respective owner/s. In no way does Jennifer-thomas claim ownership or responsibility for such items, and you should seek legal consent for any use of such materials from its owner.


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TEMPE, AZ--(Marketwired - May 9, 2014) - Tranzbyte Corporation (OTC Pink: ERBB) (
"Our name is changing to reflect the company's primary effort to maintain its leadership in the rapidly emerging Cannabis market. American Green has an opportunity to help guide both the growth and the narrative of this fast-expanding market, and to contribute to the education of the general public regarding the vast economic, medicinal, or adult-use benefits found in cannabis' many forms that are now available to Americans in over 20 states," says Tranzbyte COO Stephen Shearin.
Tranzbyte Corporation will be "pushed down" into the company structure as a subsidiary of American Green where it will continue its focus on selecting development stage companies for acquisition and possible spin-outs in the future. "Far from departing from our technology roots, Tranzbyte will remain as a division continuing to supply technological efficiencies for businesses everywhere, and, of course, remain focused in finding and acquiring companies that represent a great fit for our company," adds David Gwyther, Tranzbyte's president.
The company also announced today that it has launched its newest website at www.americangreen.com, now available for viewing beginning today. In addition, the company owns two additional websites -- www.americangreenclothing.com, which features a full line of the American Green clothing and merchandise line now available to the public, and www.americangreenzine.com, where visitors can learn of the latest in medical and adult-use cannabis news around the world. Several other web properties and media efforts are currently in production by the Company, and will be announced as they come online.
Tranzbyte encourages all folks wishing to follow the progress of Tranzbyte, American Green™, and its other subsidiaries and divisions to visit the Tranzbyte website at www.tranzbyte.com or the American Green website at www.americangreen.com and sign up to receive our 'Email Alerts' which are sparingly sent to interest-holders periodically. Just click on the orange 'contact' button after arriving at the Tranzbyte website or the "Contact" tab on the American Green website. Mobile users can also sign up by navigating to Tranzbyte's website on their android smart phone or iPhone."
Finally, shareholder and other interest-holders may track the progress of our fully automated ZaZZZ™ Machine and our entire product line by following us on Twitter @Tranzbyte or @American__Green (two underscores.)
The Tranzbyte Corporation (www.tranzbyte.com), soon to be a division of American Green, Inc, continues its plan to acquire, hold, or spin out successful divisions in what has been described in previous news releases as "dividend farming," Companies that qualify and decide to become public on their own will agree to carve out shares for Tranzbyte and dividends for their ERBB shareholders.
Tranzbyte also houses the company's 10-year-old technology division which is actively engaged in the sale of its optical media enhancement products to potential customers in the US and Asia. Products in the Tranzbyte division include FLASHAlbum™ and FlixStix -- technologies that the company plans to be incorporated into the Company's ZaZZZ Machine whiile enabling distributors of optical media (CDs, DVDs, etc.) to consolidate the best features of each medium onto a single content-protected USB flash drive.
One Bode was founded in 2006 by a group of health conscious professionals with backgrounds in sports, health, and nutrition who recognized the need for supplements to compliment any lifestyle. Focusing on plant-based nutrients and enzymes, One Bode created an assortment of products that enable the everyday person interested in improving their health to access the kind of nutritional supplements previously enjoyed by professional athletes, entertainers, and those with the time to devote their entire lives to the pursuit of such diets.
The company is combining its successful product rep program with an aggressive online campaign focusing on a realistic plan to increase sales measurably within the next 90 days of operation and throughout the coming years. The versatility of the products combined with the various dietary deficiencies present in practically everyone enables individuals to tailor a custom program designed to fit their specific needs; as such the company sees a virtually unlimited market-space for expansion.
American Green, formally Altitude Organix, became the first publicly traded medical marijuana dispensary brand in the world as American Green Corporation. Beginning in 2009, the company has developed retailing, branding, and commercial cultivating strategies in conjunction with its licensed medical marijuana retail dispensaries operating under the Altitude Organic Medicine brand name. While the Company is no longer associated with the Altitude Organic Medicine outlets in Colorado, it has spent the last year developing an "automated dispensary" that will offer the company's proprietary products to qualified adults in the US and around the world under its "American Green" brand. The Company's newly acquired YO! Debit Card has been rebranded as "ZaZZZ" for use in its proprietary machines and will be accepted at participating dispensaries and other select locations who become members of the ZaZZZ Network.
Founded in 2006, Applied RFID and its operating subsidiaries (www.proximarf.com), have a versatile and leading-edge portfolio of RFID reader, sensor tag and data logging products based on the ISO 15693 standard of high frequency (13.56Mhz) RFID.
Once the acquisition agreement with Tranzbyte is finalized, the company has plans to increase its network of global distributors and value-added resellers with plans to incorporate the technology as a tracking system for legal marijuana movements. With its "Proxima RF" line of RFID readers, sensor tags, sensor probes and data logging products, the Company seeks out opportunities with partners who have a unique software offering within market verticals where the implementation of secure data collection and temperature sensing bring real and immediate ROI to users. These key markets include: food safety, cold chain logistics and healthcare/pharmaceutical as well as factory and field maintenance applications.
Except for any historical information contained herein, the matters discussed in this press release contain forward-looking statements that involve risks and uncertainties, including those described in the Company's Securities and Exchange Commission reports and filings. Certain statements contained in this release that are not historical facts constitute forward-looking statements, within the meaning of the Private Securities Litigation Reform Act of 1995, and are intended to be covered by the safe harbors created by that Act. Reliance should not be placed on forward-looking statements because they involve unknown risks, uncertainties and other factors which may cause actual results, performance or achievements to differ materially from those expressed or implied. Forward-looking statements may be identified by words such as estimates, anticipates, projects, plans, expects, intends, believes, should and similar expressions and by the context in which they are used. Such statements are based upon current expectations of the Company and speak only as of the date made. The Company undertakes no obligation to update any forward-looking statements to reflect events or circumstances after the date on which they are made.


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Follow CBSMIAMI.COM: Facebook | Twitter
MIAMI SPRINGS (CBSMiami) – A memory of a war hero will live-on in the hallways of Miami Springs Senior High School.
Private First Class Bruce Wayne Carter, a Miami Springs Senior High alumni, was honored by way of a plaque and a ceremony held Wednesday for his courage and commitment to our country.
"I won't forget him and I won't let anybody else forget him and that's what I've done all these years is keep his name alive," said Georgie Carter Krell, Carter's mother.
During the Vietnam War in August of 1969, Carter sacrificed his own life and threw himself onto a grenade to save the soldiers standing around him.
Click here to watch Gaby Fleischman's report.
"It was a crushing blow to say the least. To lose my brother, my big brother, but it's what he wanted to do to help our country," said Cheryle Carter Nevins, Carter's sister.
After Private First Class Carter's death, he received the Congressional Medal of Honor.
He is the only soldier in Miami-Dade County to receive that recognition.
Miami's VA Hospital is also named after him.
"It's a pride and it's changed my life," his mother said.
The plaque dedicated in Carter's honor will serve as a symbol for Miami Springs students to truly understand his sacrifice.
"In a day and age where heroes are lacking this is someone the students can really look up to as someone who committed themselves to their country," said Miami Springs High School Principal Edward Smith.
Leslie Perez, a student at the school, said she feels honored that Carter attended her same school.
"I feel very honored to be here at our school and be a Golden Hawk and walk the hallways that he once walked," Perez said.
During the ceremony, the Mayor of Miami Springs also made June 3 Bruce Wayne Carter day.


text: I also meant to add, it was very noticeable when I was operating as VP2EAG from Anguilla, both with the Arrow and HT on UO-14 (sobsob) and with the dish on AO-40 S-band. The first pass on AO-40 was from a hill on the island, with the 18" DSS dish upside down on the windshield/hood to get down to the 6 degree elevation the sat was to the west. I noticed a big signal bump approaching the horizon, and a bit past my predicted LOS, even on 2.4 GHz. Great memories of that, and working from neighboring islands on AO-27 and UO-14, and running dozens of EU stations at 3AM on U/S, even split, spreading them out over 20-30 kHz. 73, Drew KO4MA