Category Archives: Texas Stem Cells

The government of Texas will allow clinics across the state to market unapproved stem-cell therapies, in a move that has met with criticism from bioethicists.

Last week Governor Greg Abbott signed off on the new legislation that allows clinics to by-pass FDA approval for investigational stem cell treatments for patients with certain severe chronic diseases or terminal illnesses. Like right to try laws in other States, the Texas legislation will give desperate patients access to therapies that provide hope after traditional medical treatments have failed.

Currently, most patients wishing to have stem-cell therapy have to travel out of the country to receive it. The new law will allow people with severe chronic or terminal illness to be treated at a clinic that purports to isolate therapeutic stem cells from adult tissuesuch as a patients own fatif their doctor recommends it after considering all other options, and if its administered by a physician at a hospital or medical school with oversight from an institutional review board (IRB). It also requires that the same intervention already be tested on humans in a clinical trial.

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The law sanctions a much broader set of therapies than federal rules, which already exempt certain stem cell interventions from FDAs lengthy approval process, provided the cells are only minimally manipulated and perform the same function they normally have in the body.

Bioethicists have expressed their concern at the move, which they say puts patients at risk of the effects of dangerous, untested therapies.

University of Minnesota bioethicist Leigh Turner said he was sceptical about whether the clinics would be adequately monitored, while NYU Langone Medical Center bioethicist Beth Roxland said it was insufficient to have the therapies tested in clinical trials while by-passing FDA approval. You could gain access to something [as long as its] being studied in a human somewhere on the planet, Roxland told Science, which in the stem cell area makes it really very scary.

LifeNews Note: This appeared at Bioedge.org and is reprinted with permission.

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Dear Colleagues and Students,

We cordially invite you to attend the 7th International Conference on Stem Cells and Cancer (ICSCC-2016): Proliferation, Differentiation and Apoptosis, Goa, India from 21-23 October 2016.

Registration and abstract submission is open now, please click on the Registration or Abstracts menu above to know more.

Conference Starts at: 9:00am, 21 Oct 2016 Conference Ends at: 6:30pm, 23 Oct 2016

Venue: Ravindra Bhavan, Margao, Goa, (www.ravindrabhavanmadgao.org)India.

You can also register and attend the conference without submitting an abstracts.

We look forward to welcoming you to the 7th ICSCC-2016 in Goa, India.

Organizer: Dr. Sheo Mohan Singh MSc(UK), PhD(Germany), PDF(USA,UK) Director, International Center for Stem Cells, Cancer and Biotechnology (ICSCCB), Pune, India (http://www.icsccb.org/)

Co-organizers: Dr. Keith Humphries Director, Terry Fox Laboratory,BC Cancer Agency, Vancouver, Canada

Dr. Christian Buske Director, Institute of Experimental Cancer Research,Comprehensive Cancer Center, University Hospital Ulm,Ulm, Germany

Dr. Laxman Gangwani Associate Professor, Department of Biomedical Sciences,Texas Tech University Health Sciences Center,El Paso, USA

Dr. Rajani Kanth Vangala Director of Research, Thrombosis Research Institute,Bangalore,India

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7th International Conference on Stem Cells and Cancer ...

http://www.naturalnews.com/037148_chemotherapy_stem_cells_regeneration.html

In one of the studies published in the journal Nature, researcher Luis Parada from the University of Texas (UT) Southwestern Medical Center in Dallas and his colleagues decided to investigate how new tumors are able to re-grow after previous ones have been wiped out with chemotherapy. To do this, Parada and his team identified and genetically labeled cancer cells in brain tumors of mice before proceeding to treat the tumors with conventional chemotherapy.

What they discovered was that, although chemotherapy appeared in many cases to successfully kill tumor cells and temporarily stop the growth and spread of cancer, the treatment ultimately failed to prevent new tumors from forming. And the culprit, it turns out, was cancer stem cells that persisted long after chemotherapy, which quietly prompted the re-growth of new tumors later down the road.

A second study published in Nature found similar results using skin tumors, while a third published in the journal Science confirmed both of the other studies in research involving intestinal polyps. It appears as though, all across the board, cancer tumors possess an inherent ability to produce their own stem cells, which can circulate throughout the body and develop into tumors. And traditional cancer treatments do nothing to address them.

"[T]raditional (cancer) therapies like surgery, chemotherapy, and radiation do not destroy the small number of cells driving the cancer's growth," says UM's Comprehensive Cancer Center. "Instead of trying to kill all the cells in a tumor with chemotherapy or radiation, we believe it would be more effective to use treatments targeted directly at these so-called cancer stem cells. If the stem cells were eliminated, the cancer would be unable to grow and spread to other locations in the body."

Alternative cancer therapies like the Gerson therapy (http://www.naturalnews.com/Gerson_therapy.html) and Dr. Stanislaw Burzynski's antineoplastons (http://www.naturalnews.com/Burzynski.html), for instance, are already successfully treating cancers in this way. But because of heavy-handed censorship and medical tyranny, these treatments are not widely accepted, and are actually considered to be fraudulent by the U.S. Food and Drug Administration (FDA) and virtually all state and federal medical boards.

Sources for this article include:

http://www.ctvnews.ca

http://www.cancer.med.umich.edu/research/stemcells.shtml

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Why chemotherapy doesn't work - NaturalNews.com

Genesis. Author manuscript; available in PMC 2009 Jul 10.

Published in final edited form as:

PMCID: PMC2708938

NIHMSID: NIHMS128325

Department of Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, Texas

Jian Chen and Chang-Hyuk Kwon contributed equally to this work.

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC-specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand-binding domain (ERT2). In the background of the Cre reporter mouse strain Rosa26lacZ, we show that the fusion CreERT2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by -galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs.

Keywords: Cre-ERT2, nestin, neural stem cells, tamoxifen, transgenic mouse, recombination

The recognition that the adult brain retains stem cells (NSCs) has fundamentally changed our view of brain plasticity (Lie et al., 2004; Ming and Song, 2005; Zhao et al., 2008). It also raises the hope of cell replacement therapy for neurodegenerative disease (Lie et al., 2004). Adult neurogenesis in the subventricular zone (SVZ) of the lateral ventricles serves to replenish olfactory bulb (OB) interneurons via the rostral migratory stream (RMS). In the dentate gyrus, neurogenesis in the subgranular layer (SGL) generates synaptically active granule neurons and has been implicated in learning, memory and mood disorders in rodents (Li et al., 2008; Ming and Song, 2005; Zhang et al., 2008; Zhao et al., 2008). The development of conditional mutant alleles using the Cre/loxP system has permitted circumvention of early lethality observed when many genes are mutated by traditional knockout, thus offering the opportunity to study gene function with spatial control (Mak, 2007). A further refinement of this technology has been the development of inducible Cre transgenes that permit temporal control of gene recombination and inactivation (Feil et al., 1997; Hayashi and McMahon, 2002). Fusion of the Cre recombinase protein with a modified estrogen receptor ligand-binding domain (ERT2) causes sequestering of the fusion protein in the cytoplasm where it cannot mediate loxP recombination. Application of estrogen or estrogen analogs, however, causes translocation of the Cre-ERT2 fusion protein to the nucleus where recombination can then be achieved.

To achieve temporal ablation of genes in the neural stem cell lineage, we have constructed a tamoxifen-inducible Cre transgene that is regulated by the neurogenic lineage specific promoter/enhancer of the nestin gene. Nestin is an intermediate filament protein specifically expressed in neural stem/progenitor cells in both developing central nervous system and adult brain. The regulatory element driving neural-specific nestin expression has been mapped to the second intron of the nestin gene (Lendahl et al., 1990; Zimmerman et al., 1994). As detailed in our studies, we show that the transgene is silent in the absence of estrogen analog. Upon activation, the expression is robust and recombination is elicited primarily in the principal neurogenic niches. Additional expression is confined to the cerebellum, certain peripheral nerves, and to the piriform cortex, a potentially novel site of neurogenesis.

The Cre-ERT2 cDNA was placed under the control of a 5.6 kb rat nestin 5 regulatory element and followed by the 668 bp of inversed nestin second intron (). Six transgenic lines were obtained after pronuclear injection and four underwent germline transmission. To assay Cre recombinase activity after induction, we crossed the CreERT2 lines with Rosa26-stop-lacZ (Rosa26lacZ) reporter mice. The Rosa26lacZ mice require Cre-mediated recombination for -galactosidase gene activation due to a stop cassette flanked by loxP sites upstream of the lacZ gene. To assess inducibility of the Cre transgene, sunflower oil vehicle (150 l) or the estrogen analog tamoxifen (1 mg) was injected into pregnant mice at embryonic day 12.5 (E12.5) and the embryos were dissected out at E14.5 for whole mount X-gal staining. In a Rosa26lacZ reporter background, exposure of the four transgenic lines to tamoxifen revealed that only two of the lines (Line 8 and Line 73) exhibited recombination activity ( and not shown). Moreover, comparison of Cre activity upon induction was similar although Line 8 was leaky, having minor but detectable Cre activity in the absence of tamoxifen. In contrast, Line 73 (Nes73-CreERT2) showed no signs of Cre activity in the absence of tamoxifen and the blue X-gal staining was found predominantly in embryonic brain and spinal cord where most nestin-positive neural progenitors are located ().

Transgene construct and tamoxifen inducibility. (a) Structure of the Nestin-CreERT2 transgene consisting of the rat nestin promoter/enhancer, cDNA encoding the CreERT2 fusion protein and inversely oriented Nestin second intron. (b) Transgene induction ...

The temporal control of Cre activity allowed us to induce Cre-mediated recombination for the purpose of tracing NSCs and their progeny at various time points. The pattern observed upon embryonic induction closely reflected the course of brain development. Tamoxifen induction at E13.5 labeled almost the entire cortex in the forebrain as well as the entire cerebellum including neurons and glia (). This coincides with the initiation of neural progenitor migration that contributes to different cortical layers in embryonic neural development (Sun et al., 2002). Induction at E17.5, when neurogenesis in the forebrain reaches completion, resulted in labeling of only the outer most layers of the cortex (), which stands in line with the inside-out pattern of cortex layer formation (Sun et al., 2002). Additionally, the thalamus and hindbrain were labeled at this time point. In the neonatal mouse brain, there is persistent mild but widespread lacZ activity, indicative of residual but rare progenitor cells throughout the parenchyma (). The most active neurogenic region at this time is the cerebellum (Herrup and Kuemerle, 1997), which showed intense lacZ staining following induction at E17.5 through P7 (). Mouse cerebellum development is considered to be complete by 3 weeks after birth, however our Nes73-CreERT2;Rosa26lacZ mice showed strong Cre activity in the anterior part of cerebellum when induced 4 and 8 weeks after birth (, and ; and see below). Nonetheless, in the anterior brain, by 4 weeks of age the SVZ and SGL are the most neurogenic regions as assayed by tamoxifen-induced Cre activity ().

Novel Cre activity. Nes73-CreERT2;Rosa26lacZ mice were treated with tamoxifen at 4 weeks of age and analyzed at 8 weeks (ac, eh, left and right panel of i). Abundant -Gal expression was detected in the anterior part of cerebellum ...

Adult NSCs modify their gene expression as they migrate and differentiate. In the SVZ, glial fibrillary acidic protein (GFAP) positive cells are considered to be stem cells (Doetsch et al., 1999). When differentiation starts and neuronal fate of the progenitor cells has been specified, cells begin to express doublecortin (DCX) and migrate into the OB through the RMS to finally become NeuN-positive mature neurons (Doetsch et al., 1999; Ming and Song, 2005). To determine the sites of primary Cre recombinase activity, we examined the SVZ of 4-week-old Nes73-CreERT2;Rosa26lacZ mice 48 h after a short pulse of tamoxifen, since both GFAP-positive neural stem cells and some transient amplifying progenitor cells express nestin. X-gal staining followed by immunohistochemistry (IHC) with GFAP or DCX antibody revealed that the majority of Cre activity resides in GFAP-positive SVZ cells close to the lateral ventricle, with only rare DCX-positive SVZ or RMS cells showing recombination (). This was further confirmed using an estrogen receptor antibody to show double labeling of Cre-ERT2-positive cells with the stem cell marker GFAP, and with S100, a marker of radial glia-derived ependymal cells (Supp. Info. Fig. 1) (Spassky et al., 2005). These studies indicate that the primary site of tamoxifen-activated Cre recombinase is the GFAP-positive, SVZ stem cell population.

Cre activity in adult NSC niches and migration targets. (a) Representative X-gal stained brain sections from mice 48 h after two tamoxifen administrations at P28 (12-h interval). X-gal signal was mainly restricted to SVZ (a1), with little or no signal ...

To measure the efficiency of tamoxifen-induced recombination in our Nes73-CreERT2 mice, we crossed them with the Rosa26YFP reporter line to generate Nes73-CreERT2;Rosa26YFP mice and then induced these mice with tamoxifen at 4 weeks of age. We then harvested brain sections from the induced mice at 6 weeks of age, and performed immunofluorescent double-labeling with GFP and Sox2 antibodies (Supp. Info. Fig. 2). The percentage of GFP/Sox2 double-positive cells divided by the number of Sox2 positive cells in the SVZ was used to determine recombination efficiency. This quantification analysis revealed that 75 4% of Sox2-positive cells in the SVZ have been targeted 2 weeks after a 5-day tamoxifen induction.

To further study the dynamics of stem/progenitor cell migration and differentiation, Nes73-CreERT2;Rosa26lacZ mice were induced at 4 weeks of age and examined by X-gal staining 2 or 4 weeks later ( and ). The dynamics of Cre-active cells in the hippocampus over time was not very dramatic (), however in the SVZ, an increase in the number of Cre active cells in an expanded ventricular area was evident 4 weeks after induction (). These results suggest a precursor-progeny relationship in which, after 2 weeks of induction, a significant number of new progenitor cells have been generated by stem cells and are beginning to disperse from the SVZ. Similarly, in the OB 2 weeks after induction, the X-gal positive cells were confined to a central cluster, whereas 4 weeks postinduction the cells were dispersed throughout the OB (). We interpret this result to indicate that at 2 weeks postinduction, cells are just arriving to the OB via the RMS and are confined to this central area, whereas at 4 weeks postinduction, these labeled cells have now dispersed throughout the OB. A similar, although more restricted, migration was also observed in hippocampus, where -Gal and NeuN double-positive neurons first appear close to the SGL 2 weeks after induction but by 4 weeks postinduction have migrated deeper into the granular layer ().

To explore the identity of the Cre-active cells, immunofluorescent double labeling was used to characterize Nes73-CreERT2;Rosa26lacZ mice 4 weeks after induction (). -Gal immunoreactivity was found in nestin and GFAP-positive neural stem/progenitor cells in the SVZ and SGL (). In the anterior part of the SVZ and SGL, DCX-positive neural progenitors also showed Cre activity (). In addition, a majority of the cells in the RMS express both -Gal and DCX (). Furthermore, NeuN-positive mature neurons that also retained -Gal immunoreactivity could be found in the HP and OB (). A small number of GFAP-positive astrocytes in the OB and the corpus callosum (CC) also expressed the reporter gene -Gal (), indicating the presence of Cre activity in multiple cell types in the NSC lineage. This result is consistent with recent quantitative lineage tracing studies (Lagace et al., 2007).

The significant amount of Cre activity induced in anterior cerebellum of adult mice was unexpected (). shows a representative eight-week-old brain from a mouse that was induced with tamoxifen at 4 weeks of age. The -Gal positive cells were mostly NeuN-positive inner granular layer (IGL) granule cells and Bergmann glia that extend long processes to the surface of the cerebellum (). Consistent with previous reports that Bergmann glia express NSC markers such as nestin and Sox2 (Mignone et al., 2004; Sottile et al., 2006), we found that Cre-active Bergmann glia also expressed the NSC marker nestin (). However, the Cre-ERT2 fusion transgene was also expressed in some Sox2-negative cells in the IGL (, middle panel), suggesting potential aberrant expression of the Nestin-CreERT2 transgene. Mild but reproducible tamoxifen-induced Cre activity was also observed in the piriform cortex (), which has also been reported to be a potential neurogenic region (Pekcec et al., 2006). We next assessed tamoxifen-induced Cre activity in other regions using whole mount X-gal staining, and found that the dorsal root ganglia (DRG) but not the spinal cord showed Cre activity (). Histologic examination revealed that less than half of the DRG neurons undergo Cre-mediated recombination (). In addition, Cre activity was detected in the optic nerve and trigeminal ganglia in mice induced at neonatal (, middle panel) or adult stages (, right panel). Collectively these data indicate that the nestin promoter/enhancer employed to generate this tamoxifen inducible transgene, exhibits remarkable fidelity to the endogenous neural expression with only a few potential sites of discrepancy.

Detailed analysis of traditional Nestin-Cre transgenic lines has revealed Cre activity outside the CNS, for example, in the kidney and in somite-derived tissues (Dubois et al., 2006). To determine whether Cre activity in the Nes73-CreERT2 mice was restricted to the nervous system, Nes73-CreERT2;Rosa26lacZ mice were induced for 5 days starting at P0 and analyzed at 8 weeks of age by whole-mount X-gal staining of internal organs including the heart, lung, liver, thymus, spleen, kidney, pancreas and stomach. With the exception of the esophagus, where neonatal but not adult exposure to tamoxifen induced Cre activity (, Supp. Info. Fig. 3) and stomach, where spontaneous lacZ activity is present in controls (, Supp. Info. Fig. 3) (Kwon et al., 2006), we found no evidence of obvious reporter expression in the absence or presence of tamoxifen (see ).

Cre activity is not observed in internal organs. Nes73-CreERT2;Rosa26lacZ mice were treated with vehicle (Veh) or tamoxifen (Tmx) at P0 for 5 days. Different organs were then dissected out at 8 weeks and subjected to whole mount X-gal staining. Endogenous ...

The rediscovery of neurogenesis in the adult brain has led to reawakened interest in the role of new neurons in the mature brain. The SVZ is a major site of neurogenesis for OB interneurons, although emerging evidence suggests additional roles. In the hippocampus, neurogenesis has been implicated in mood modulation and in learning and memory (Li et al., 2008; Lie et al., 2004; Zhao et al., 2008). On the dark side, stem/progenitor cells in the CNS have been implicated as the source of glioblastoma (Kwon et al., 2008; Sanai et al., 2005; Zhu et al., 2005). Specific ablation or activation of genes implicated in hippocampal function and in glioma can be achieved with our tamoxifen-inducible Cre transgene and we have developed successful models of both SVZ stem/progenitor cell-dependent induction of glioma and hippocampal stem/progenitor cell-dependent antidepressant insensitive mice using this tamoxifen-inducible Cre mouse line (Li et al., 2008; Llaguno et al., submitted).

Still, there is much to be learned about the precise role of neural stem cells in normal brain function and in associated pathologies. For example, in this report we describe novel sites of nestin-Cre recombinase activity. Whether this activity identifies previously undetected sites of neurogenesis or simply ectopic Cre expression remains to be rigorously determined. Of note, a second, independently derived transgenic line, Nes8-CreERT2, shows a similar pattern of inducible expression (data not shown) leading us to favor the conclusion that the expression outside the SVZ and SGZ is not due to position effects at the site of transgene insertion but rather is a reflection of the properties of the transgenic construct. Stem cells have been isolated from neonatal cerebellum and they are reported to be prominin/CD133-positive and Math1-negative (Klein et al., 2005; Lee et al., 2005). We observe Cre activity in the cerebellum from E17.5 through 8 weeks of age. Although diminishing over time, a clear gradient is observed that becomes progressively more anterior. The lacZ positive cells resulting from activation of the Rosa26 reporter possess the characteristic morphology of granule cells. In adult cerebellum, the Bergmann glia retain a morphology reminiscent of radial glia which can generate neurons and adult NSCs during brain development (Gotz and Barde, 2005; Merkle et al., 2004). In addition, Bergmann glia still express stem cell markers such as Sox2 and nestin (Mignone et al., 2004; Sottile et al., 2006). On the other hand, only rarely have cells with BrdU incorporation been observed in adult cerebellum, even after growth factor infusion (Grimaldi and Rossi, 2006). We also found that a number of cells in the anterior cerebellum targeted 2 days after acute tamoxifen administration were positive for NeuN but not GFAP or nestin (Supp. Info. Fig. 4), suggesting that the cre activity in the IGL was more likely due to promoter leakiness (Supp. Info. Fig. 4). Further study is needed to resolve this issue.

A series of similar inducible Nestin-Cre transgenes has recently been reported, although the extent of expression over time and expression outside the nervous system was not described (Supp. Info. Table 1) (Balordi and Fishell, 2007; Burns et al., 2007; Imayoshi et al., 2006; Kuo et al., 2006; Lagace et al., 2007). Eisch and co-workers recently described a tamoxifen-inducible Cre transgenic mouse line with no obvious Cre activity in the cerebellum upon tamoxifen induction (Lagace et al., 2007). The fact that our transgenic construct included only intron 2 of the nestin gene whereas their construct contained nestin exons 13 could account for this discrepancy (Zimmerman et al., 1994). It is possible that our more limited nestin construct might lack cerebellar-specific repressor sequences. Another potentially significant variation is the use of a Rosa26lacZ reporter line versus the Rosa26YFP reporter used by Lagace et al. (2007). Both the sensitivity of the reporter and perhaps the recombinogenic efficiency could in principle differ, leading to these discrepancies. We also observe Cre activity in the adult piriform cortex. This is in accordance with previous reports of BrdU incorporation in this region, leading to the suggestion of additional neurogenic niches (Pekcec et al., 2006).

We examined our mice for leakiness as well as for inducible transgene expression in the peripheral nervous system (PNS) and multiple organs. In contrast to many other Nestin reporter transgenic mice (Day et al., 2007; Dubois et al., 2006; Gleiberman et al., 2005; Li et al., 2003; Ueno et al., 2005), we found no evidence of obvious leakiness or of inducible transgene activation outside the CNS except in the PNS, where inducible expression was found both in the DRG and trigeminal ganglion, and in the esophagus. It is possible that our Nestin-CreERT2 transgene has a more restricted expression pattern or that the tamoxifen induction efficiency is lower in certain tissues. In addition, whole mount X-gal staining of the organs makes it difficult to capture rare Cre-positive cells if they do exist. DRG have been used to culture neurospheres (Li et al., 2007), and it will be of interest to determine whether our transgene is active in these progenitor cells, which would provide supportive evidence for the existence of additional neural stem/progenitor niches. Subsequent detailed lineage tracing of the Cre expressing cells will more clearly address this issue.

A 2.0 kb fragment of CreERT2 and SV40 polyA sequence of the pCre-ERT2 vector (Feil et al., 1997) were amplified using a PCR technique that also generated 5 Not1 and 3 Spe1 sites. After enzymatic digestion, purified fragment was ligated to an 8.9 kb fragment from pNerv (Panchision et al., 2001; Yu et al., 2005) digested with Not1 and Xba1. The resulting pNes-CreERT2 construct contains a 5.6 kb rat nestin 5 genetic element from pNerv, a 2.0 kb CreERT2 and SV40 polyA sequence from pCre-ERT2 and a 668 bp of reversed second intron of rat nestin from pNerv (). After Sal1 digestion, an 8.3 kb band was purified and microinjected into the pronuclei of fertilized eggs from B6D2F1 mice. Among 28 pups born after two rounds of transgenic injection, six contained the transgene, and four of them transmitted to germline. Rosa26lacZ mice were obtained from Jackson Laboratories (Bar Harbor, ME), Rosa26YFP mice were kindly provided by Dr. Jane Johnson. All the mice were maintained in a mixed genetic background of C57BL/6, SV129 and B6/CBA. Nestin73-CreERT2; Rosa26lacZ mice were generated by crossing male Nestin-CreERT2 mice with female Rosa26lacZ mice. Genotyping of the mice was performed as described previously (Kwon et al., 2006). All mouse protocols were approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center.

Tamoxifen (Sigma-Aldrich, St. Louis, MO) was dissolved in a sunflower oil (Sigma-Aldrich, St. Louis, MO)/ethanol mixture (9:1) at 6.7 mg/ml. For initial screening of the embryonic induction of the transgenic lines, 150-l tamoxifen (1 mg) or vehicle (sunflower oil/ethanol mixture only) was injected intraperitoneally into pregnant mice at embryonic day E12.5 (E12.5 hereafter). Embryos were dissected out 2 days later and subjected to X-gal staining. For in utero induction, 150-l tamoxifen (1 mg) or vehicle was injected intraperitoneally into pregnant mothers at E13.5 or E17.5, and pups were analyzed 1 month after birth. For neonatal induction, 12.5-l tamoxifen (83.5 mg/kg body weight) or vehicle per gram of mouse body weight was injected into lactating mothers (tamoxifen can be delivered to pups through the mothers milk) at P0 or P7, once a day for 5 days and the pups were analyzed 4 weeks after the first induction. For induction in adult mice, 12.5-l tamoxifen (83.5 mg/kg) or vehicle per gram of body weight was injected intraperitoneally into 4- or 8-week-old mice twice a day for five consecutive days and then analyzed 2 or 4 weeks after the first induction.

Mice were dissected and perfused as previously described (Kwon et al., 2006). For whole mount X-gal staining, the embryos or organs were carefully dissected out, washed with phosphate-buffered saline (PBS), and then fixed in 2% (w/v) paraformaldehyde (PFA; in PBS) for 1 h at 4C. Postnatal brains were postfixed in 2% PFA overnight (O/N) at 4C, embedded in 2.5% chicken albumin sagittally or coronally, and then cut into 50-m thick sections by vibratome (Leica, Nussloch, Germany). Every fifth sagittal section or 12th coronal section was chosen to perform X-gal staining and comparable sections were selected for further immunostaining according to the X-gal staining result. X-gal staining of organs and sections was performed as described (Kwon et al., 2006).

Four Nestin73-CreERT2;Rosa26YFP mice were induced at 4 weeks of age as described above and perfused with 2% PFA at 6 weeks of age. The brains were dissected out, postfixed in 4% PFA O/N at 4C, processed and embedded in paraffin blocks. Five-m thick sagittal sections were cut until the lateral ventricle was gone. H&E staining was performed on every fifth slide to determine comparable sections. Every 10th of comparable sections was subjected to GFP (Aves Labs, Tigard, OR) and Sox2 (Chemicon, Temecula, CA) immunofluorescence staining, and three random regions of the frontal SVZ of each section were selected for counting. The efficiency was determined by the percentage of GFP (mean 203)/Sox2 (mean 270) double-positive cells out of the total Sox2-positive cells in SVZ.

Free-floating immunofluorescence staining was performed on 50-m thick sections. Antibodies used for the staining were against -galactosidase (ICN, Aurora, OH), GFAP, nestin (BD Biosciences, Bedford, MA), doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA), NeuN (Chemicon, Temecula, CA), Mash1 (BD Biosciences, Bedford, MA), S100 (Sigma-Aldrich, St. Louis, MO). Alexar-488 or Alexar-555 conjugated goat anti-mouse or anti-rabbit (Molecular Probes, Eugene, OR) and Cy2 or Cy3 donkey anti-goat, anti-rabbit antibodies (Jackson Immunoresearch, West Grove, PA) were used to visualize primary antibody staining. Images were taken on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany). For ER and Sox2 staining, 5-m thick paraffin sections were first stained with estrogen receptor antibody (Lab Vision, Fremont, CA) and visualized by DAB substrate with nickel solution (Vector Laboratories, Burlingame, CA). The slides were then washed with PBS three times, stained with Sox2 antibody (Chemicon, Temecula, CA), and visualized by Vector NovaRED (Vector Laboratories, Burlingame, CA). Images were taken with a Nikon 2000 CCD camera (Nikon, Japan). All images were assembled using Adobe Photoshop CS and Illustrator CS (Adobe Systems Incorporated, San Jose, CA).

We thank Steven Kernie for providing pNerv plasmid, Jane Johnson and Frank Costantini for providing Rosa26YFP mice, Steven McKinnon, Shirley Hall, and Linda McClellan for technical assistance, Renee McKay for reading the manuscript, and Jane Johnson, James Battiste, Jing Zhou, and Yun Li for discussion and suggestions.

Additional Supporting Information may be found in the online version of this article.

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Inducible Site-Specific Recombination in Neural Stem ...

Mesquite TX Stem Cell Research is a complex and beneficial science using stem cells in a lab environment to better understand how normal human development works, and also to look for and develop new treatments for a wide range of human ailments. Mesquite Texas Stem Cell Research involves two types of stem cells, classified as either embryonic stem cells or adult stem cells, which are used according to the type of Mesquite TX Stem Cell Research that is desired.

Embryonic stem cells are derived from pre-embryos, called blstocysts, approximately three to five days old. They are created specifically for fertilization treatments in the Mesquite Texas Stem Cell Research lab, will not be used to start a pregnancy, and will be discarded if not used for research. Doctors use in-vitro fertilization to create an embryo in a culture dish, which after three to five days becomes a blstocysts. Mesquite TX Stem Cell Research lab technicians then extract the inner cell mass from the blstocysts, which is used to derive embryonic stem cells in the Mesquite Texas Stem Cell Research facility.Embryonic stem cells are classified as pluripotent.

This means they can develop into any type of cell in a fully developed human body. It should be noted that embryonic stem cells cant develop into placenta or umbilical cord tissues, but they do appear to be able to develop into any other type of cell in a human body. What is so important about embryonic Mesquite TX Stem Cell Research is that it enables very flexible research, as the stem cells can be grown into any type of cell needing to be researched, at any time, at the Mesquite Texas Stem Cell Research facility. This makes for more efficient and more productive stem call research, promising a faster path to cures for ailments that devastate humanity. Mesquite TX Stem Cell Research cannot use adult stem cells to generate just any desired tissues since they are already programmed. They are quite useful nonetheless, and Mesquite Texas Stem Cell Research doctors have identified caches of adult stem cells in several tissues of the human body.

Mesquite TX Stem Cell Research in general has been able to make some wonderful advancement and create excellent treatments using adult stem cells. But there are limitations to doing Mesquite Texas Stem Cell Research using "only" adult stem cells. Adult stem cells are able to give rise to related kinds of cells in their home tissues, but for example Kidney stem cells cannot generate heart cells, and liver stem cells cannot generate brain cells.

A great deal of Mesquite TX Stem Cell Research remains to be done, and at this point Mesquite Texas Stem Cell Research doctors have developed a technique for getting an adult stem cell to behave similar to an embryonic stem cell. This specialized Mesquite TX Stem Cell Research technique creates what are called induced pluripotent stem cells (iPS). They can be produced from adult cells in skin, fatty tissue, and other sources. With this, Mesquite Texas Stem Cell Research remains a promising field. There is of course a great deal more work to do, but Mesquite TX Stem Cell Research promises to benefit mankind in many profound ways.

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Mesquite Texas Stem Cell Research | Mesquite TX Stem Cell ...

University of Texas M. D. Anderson Cancer Center

IMAGE:This is Jeffrey Molldrem, M.D. view more

Credit: MD Anderson Cancer Center

The University of Texas MD Anderson Cancer Center and Astellas Pharma Inc. have signed an option agreement to research and develop a new treatment for patients with acute myeloid leukemia (AML).

The collaboration grants Astellas an option to firstly negotiate an exclusive, worldwide license at the end of Phase Ib, with both Phase Ia and Phase Ib studies to be conducted by MD Anderson. The agreement also includes up to $26 million as an option premium and for research and development funding.

The collaboration will focus on h8F4 technology, a humanized monoclonal antibody invented by Jeffrey Molldrem, M.D., professor of Stem Cell Transplantation and Cellular Therapy at MD Anderson. The antibody h8F4 targets an HLA-restricted peptide called PR1/HLA-A2, which is expressed in cancer cells and cancer stem cells. Molldrem will lead these research efforts with Carlo Toniatti, M.D., Ph.D., executive director of MD Anderson's Oncology Research for Biologics and Immunotherapy Translation (ORBIT) platform.

"Current treatments for aggressive leukemias are often toxic," said Molldrem. "We desired to develop a safer, yet more potent, therapy for these aggressive cancer types that currently have poor survival outcomes. Unfortunately, advancing novel discoveries from the laboratory to drug development has been historically challenging. We hope that this important collaboration will allow us to deliver much-needed antibody-based treatment to the patient's bedside more quickly."

"h8F4 has a radically novel anti-tumor activity and this collaboration provides MD Anderson and Astellas with a great opportunity to potentially deliver a first-in-class antibody drug to patients with AML," commented Yoshihiko Hatanaka, president and CEO of Astellas. "Astellas continues to focus on developing novel therapies in areas of unmet medical need through in-house development and external collaborations."

While monoclonal antibodies are very common in oncology, generating antibodies against HLA-restricted peptides has proven difficult. To develop viable antibody drugs, MD Anderson created ORBIT for its Moon Shots Program to centralize this type of research. The program is an ambitious initiative to accelerate the conversion of scientific discoveries into clinical advances and significantly reduce cancer deaths.

"This is an outstanding addition to MD Anderson's Moon Shots Program to deliver accelerated solutions for cancer treatment," said Ronald DePinho, M.D., president of MD Anderson. "These are exciting times for cancer drug development and I'm proud that eminent scientists like Drs. Molldrem and Toniatti are leading the way. While it's true that myeloid cancer has not responded well to standard therapies, this novel solution looks promising."

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MD Anderson, Astellas Pharma sign option agreement for monoclonal antibody drug targeting acute myeloid leukemia

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Newswise DALLAS March 6, 2015 The Cancer Prevention and Research Institute of Texas (CPRIT) has awarded UTSouthwestern Medical Center researchers more than $7.5 million in research grants to improve diagnostic and therapeutic services and research relating to cancers of the brain, breast, throat, and bone, as well as to improve scientific understanding of cancer biology.

UTSouthwestern received an additional $4 million for recruiting emerging cancer scientists. The February awards bring the total awarded by CPRIT to UTSouthwestern for cancer research, recruitments, and prevention efforts to $34 million in fiscal year 2015, more than any other Texas institution.

New research grants were awarded to seven UTSouthwestern researchers at the Harold C. Simmons Comprehensive Cancer Center as part of CPRITs Individual Investigator Research Awards.

The projects by these researchers underscore the vitality of the collaborative effort we value and encourage at the Simmons Cancer Center, and the diversity of approaches that are needed to expand opportunities for those facing a cancer diagnosis, said Dr. James Willson, Associate Dean of Oncology Programs at UTSouthwestern, and Professor and Director of the Simmons Cancer Center, the only National Cancer Institute-designated cancer center in North Texas and one of just 68 in the country. Dr. Willson holds The Lisa K. Simmons Distinguished Chair in Comprehensive Oncology.

Dr. Luis Parada, Chairman of Developmental Biology, Director of the Kent Waldrep Foundation Center for Basic Neuroscience Research, and holder of the Diana K. and Richard C. Strauss Distinguished Chair in Developmental Biology and the Southwestern Ball Distinguished Chair in Nerve Regeneration Research, was awarded $900,000 to study a small chemical compound that stops stem cell growth in gliomas (brain cancer). His lab has been instrumental in identifying molecules that inhibit nerve regeneration after injury.

Dr. Craig Malloy, Professor with the Advanced Imaging Research Center, Internal Medicine, and Radiology, who holds the Richard A. Lange, M.D. Chair in Cardiology, was awarded $897,311 in partnership with Texas A&M University to develop new technologies to study metabolism in breast cancer. This collaboration leverages the strengths in human metabolism at UTSouthwestern, electrical engineering expertise at Texas A&M, and the distinctive 7 Tesla magnetic resonance imaging (MRI) device at the Bill and Rita Clements Advanced Medical Imaging Building at UTSouthwestern. MR images at 7T allow investigators to observe small anatomical structures and to monitor tissue biochemistry without biopsies or radiation.

Dr. Zhijian Chen, Professor of Molecular Biology and with the Center for the Genetics of Host Defense, who holds the George L. MacGregor Distinguished Chair in Biomedical Science, was awarded $889,185 to study how to improve cancer immunotherapy through a pathway called the Cytosolic DNA Sensing Pathway. The pathway induces type-I interferons, which are important for activating cytotoxic T cells to kill tumors.

Dr. Steve Jiang, Professor and Vice Chairman of Radiation Oncology, Chief of the Division of Medical Physics and Engineering, and holder of the Barbara Crittenden Professorship in Cancer Research, was awarded $858,356 to study how to take online adaptive radiotherapy technologies developed in the lab into clinical practice. Online adaptive radiotherapy allows real-time adjustments to provide individualized cancer radiotherapy that can help reduce unwanted exposure to healthy tissues.

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CPRIT Awards Faculty $11.5 Million for Recruitment and Research in Brain and Bone Cancer, Biology, and Immunotherapy

11 hours ago Bursting roundworm. Due to a defect in regulation of the let-7 target lin-41 the worm bursts and dies at the larval to adult transition. Green: seam cell nuclei, red: cell membranes.

It has generally been believed that microRNAs control biological processes by simultaneously, though modestly, repressing a large number of genes. But in a study published in Developmental Cell, a group of scientists led by Helge Grosshans have now shown that miRNAs can control the development of a roundworm through regulation of a single target.

The discovery, some 15 years ago, of the small RNA molecule let-7 opened up a whole new field of research. It became apparent that genes in a wide variety of organisms from roundworms to humans are regulated by a host of so-called microRNAs (miRNAs). In the development of the roundworm, let-7 plays a key role: if it is defective, the worm bursts and dies as a result of abnormal development of the sexual organs. Although in the meantime, much has been learned about the molecular mechanisms underlying miRNA function, it remained unclear how this dramatic effect of let-7 was to be explained.

An elegant study by a group led by Helge Grosshans at the FMI has now revealed how let-7 controls normal vulval development in the roundworm. Moreover, by showing that miRNAs can produce significant biological effects through regulation of one single target gene, these findings make a substantial contribution to current theories on the functioning of miRNAs.

It is known that miRNAs can bind many different messenger RNAs (mRNAs), thus repressing protein translation or facilitating mRNA degradation. However, as the effect is usually modest, it was assumed that the biological activity of miRNAs is mediated by coordinated repression of numerous different mRNAs.

The new study demonstrates that the dramatic effect of let-7 is attributable to interaction with lin 41 mRNA alone, although let-7 also regulates other mRNAs. These other interactions are, however, dispensable and inconsequential. Grosshans comments: "The finding that a microRNA can have such a dramatic effect through interaction with a single gene is new. What our study also shows is that it is not enough to measure the interaction of RNA molecules we need to elucidate how such interactions influence a specific function."

Here, the scientists benefited from a new genome editing technology known as CRISPR-Cas9, which allows base pairs to be replaced in a targeted fashion. As first author Matyas Ecsedi explains, "We were able to modify the binding of let-7 to specific mRNAs and then directly observe what effect this had on the development of the sexual organs." As well as providing new insights into the functioning of miRNAs, this approach has the potential to advance the safe and effective use of miRNAs for therapeutic purposes.

Evolutionarily conserved: let-7 and LIN-41

The rise of microRNA research was largely due to the fact that let-7 is evolutionarily well conserved i.e., it occurs in many different animals. It was thus clear that let-7 is not peculiar to the roundworm, but plays a role in various species. Over the years, let-7 was found to be universally important as a regulator of stem cell processes.

LIN-41/TRIM71 likewise occurs in various organisms, assuming a similar function in each case. In stem and progenitor cells, LIN-41 promotes cell division and prevents cell differentiation, while let-7 serves the opposite function and represses LIN-41. As a pair, let-7 and LIN-41 are therefore of great interest in the quest to improve our understanding of stem cell processes.

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A single target for microRNA regulation

Using engineering principles to understand the complexity of stem cells and cancer

Xiling Shen will join the biomedical engineering department in Duke Universitys Pratt School of Engineering in July 2015. An electrical circuit and computer systems expert by training, Shen applies his engineering background to understand the complex regulatory networks behind biological systems and diseases.

Medical research has long taken a reductionist approach to health care, Shen said. For example, researchers often focus on finding the culprit protein that gives rise to a disease, so that the protein can be targeted to produce a cure. Many challenging diseases such as cancer, however, are caused by complex interactions among a network of many proteins and RNAs as well as changes to their physical environmentwhich cant be fully understood outside of that context.

Xiling Shen

We need better computer and animal models that accurately reproduce the complexity of biological systems and human diseases, said Shen, who joins Duke from Cornell University. Better models can better recapitulate the physiological environments seen by our own cells, which produce better studies and results.

Shen earned his bachelors, masters and doctorate degrees from Stanford University, before conducting postdoctoral research at the University of CaliforniaBerkeley. He then joined Cornell Universitys faculty as an assistant professor of electrical and computer engineering and biomedical engineering in 2009. He also worked as an analog and wireless circuit designer at Barcelona Design and Texas Instruments between 2001 and 2004.

One example of Shens work is a recent study that revealed for the first time that small RNAs called microRNAs instruct cancer stem cells to divide in two different ways. Besides a division process that results in two cancer stem cells, his laboratory discovered that they can also divide into one cancer stem cell and one tissue cell. This allows tumors the flexibility to grow while maintaining a steady supply of stem cells. Such microRNAs can be therapeutically target to disrupt tumor growth.

Another recent study discovered that cancerous cells hijack mechanisms originating in the immune system to catch a ride to different organs during metastasis. Leveraging this discovery, Shens laboratory injected engineered human cancer cells into mouse embryos. These mice grew up recognizing human cancer cells as their own, which led to the growth of human tumors in the organs where they would naturally occur.

In the past, cancerous human cells have just been injected under the skin of mice to grow tumors for testing, said Shen, who hopes that realistic disease models will enable researchers to find better cancer drugs that have been elusive so far. But that microenvironment is completely different from those of various organs, and that can play a huge role in how well the tumor grows and how well treatments work. Genetically engineering switches into cancerous cells to direct them to the proper organ will provide a more accurate model for testing.

Shen sees the close physical proximity and institutional ties between Duke Medicine and the biomedical engineering department as a potential boon for his research. As an engineer, Shen says he isnt satisfied with simply discovering new things; he wants to take his work from bench to bedside to have a large societal impact.

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Xiling Shen: An Engineering Approach to Biomedical Challenges

13 hours ago The fluorescent probe AIE-MitoGreen-1 reveals changes in mitochondrial organization in brown adipose cells as they mature over the course of a week. Credit: Royal Society of Chemistry

A new cellular labeling strategy gives researchers an efficient tool for studying the development of tissue that could help prevent the onset of obesity and cardiovascular disease.

Most people think about fat in terms of the white adipose tissue that stores the body's excess energy, and which steadilyand visiblyaccumulates as one becomes out of shape or obese. However, there is another type of fat tissue that can prevent rather than promote weight gain. "Brown adipose tissue not only stores fats, but also has the ability to burn fats to release energy as heat," explains Bin Liu of the A*STAR Institute of Materials Research and Engineering.

Liu sees this tissue as a promising target for anti-obesity drugs, and her group set about designing a fluorescent molecule that could help scientists visualize the development of brown adipose cells. These cells can be characterized based on the number and organization of their mitochondria, the organelles that drive cellular metabolism. However, existing mitochondrial dyes tend to absorb each other's fluorescence at high concentrations, resulting in a weaker overall signal as they accumulate.

In collaboration with Hong Kong University of Science and Technology researcher Ben Zhong Tang, Liu's team devised a fluorescent dye that exhibits 'aggregation-induced emission'. "This means that the probe does not emit fluorescence in dilute solutions," explains Liu, "but it becomes highly fluorescent when it accumulates in mitochondria, without any self-quenching effects."

After 20 minutes of treatment with their AIE-MitoGreen-1 probe, Liu's group achieved bright labeling of mitochondria in brown adipose cells that lasted for more than a day. This labeling approach also left cultured cells largely unharmed, whereas only 10 per cent of cells survived prolonged treatment with a commercially available mitochondrial dye. The researchers subsequently used AIE-MitoGreen-1 to monitor the development of brown adipose tissue from precursor cells, observing changes in cell shape and mitochondrial organization over seven days (see image).

Since the basic stages of brown adipose development are well characterized, this probe could help identify treatments that stimulate or impede this process. "We hope to use our probe to monitor the activity of brown adipose cells in response to various stimuli, such as drug intervention or temperature changes," says Liu. Her group aims to further improve their probe so that it shines longer and brighter. Ultimately, she hopes to develop variants that fluoresce at near-infrared wavelengths, which can be detected deeper within living tissue. "We would apply these probes to long-term monitoring of brown adipose cells in animal models."

Explore further: New source of fat tissue stem cells discovered

More information: Gao, M., Sim, C. K., Leung, C. W. T., Hu, Q., Feng, G. et al. A fluorescent light-up probe with AIE characteristics for specific mitochondrial imaging to identify differentiating brown adipose cells. Chemical Communications 50, 83128315 (2014). dx.doi.org/10.1039/c4cc00452c

Researchers have found a new source of stem cells that produce fat tissue, findings presented today at the European Congress of Endocrinology in Wrocaw, Poland, show. This unique in vitro human stem cell model of brown ...

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Fluorescent probe for labeling mitochondria helps scientists study fat-burning brown adipose tissue