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Amniotic Stem Cell Therapy in Salt Lake City, Park City Utah

Posted: August 22, 2016 at 8:47 am

Current treatments for the painful and debilitating effects of arthritis or joint pain rely on early intervention with synthetic drugs and, in severe cases, surgery.

The human body is composed of more than 200 cell types. Each of these cells in the body is designated to perform a particular function. For e.g. its the pancreatic cells that ensure you pancreas are functioning properly. But there are some other cells in human body, which are different. These cells are called stem cells. These cells not only demonstrate their capacity to generate more stem cells but also help in making different cell types. For over a decade the adult stem cells have been used therapeutically for treating certain cancer as well as rare blood diseases.

Medical practitioners often call stem cell therapy a gift of science. So is the therapy scientifically proven type of regenerative medicine? Yes, it is! There is a considerable amount of excitement about this therapy. Stem cell has the potential to revolutionize the concept of healthcare. There is lot medical practitioners are doing with the stem cells. Diseases such as strokes and diabetes will be soon treated by the therapy. Two other areas where the therapy has worked wonders include bone and blood cancer treatment and injuries to the joint.

Next stem cell product that is making headway into the healthcare industry is for burn and wound healing. Doctors are also planning on injecting the stem cells into to brain of a patient with cerebral palsy to help enhance the function as well as restore the brain tissue. Another major field is where medical practitioners are combining 3D bio printing with stem cell regenerative medicine.

Amniotic stem cell therapy has been acknowledged in the medical history across the world as a biggest discovery in natural healing. The stem cells are first injected into the patients injured area. They then metamorphosize into different cells, when often depends upon the instructions provided by a human body. So they may become a tissue cells, bone cells or other kind. This is what medical practitioners believe is so unique about their growth. Huge number of cells injected into the patients body will help in healing the injuries at an accelerated pace. Best results are seen in neuropathy, knee pain and osteoarthritis.

Fat, cortisone and other medicines offer temporary cure. But stem cells have the capability to restore the regenerative tissue and bone back to their original condition. One of the prime components here is the high uronic acid that is known to lubricate your joints and tendons, easing pain and restoring your mobility.

The injections used in treating patients with joint problems contain naturally developing anti-inflammatory agents that actually stimulates tissue repair. Besides, there have been no cases of patient rejection recorded so far. Over 10,000 injections have been used on patients without any cases of adverse side effects. So if you want to avoid expensive and painful surgery and dependency on prescriptive medication access our Amniotic stem cell therapy and bid adieu to your pain.

*Disclaimer: These statements have not been evaluated by the FDA

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Amniotic Stem Cell Therapy in Salt Lake City, Park City Utah

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Inducible Site-Specific Recombination in Neural Stem …

Posted: August 22, 2016 at 8:47 am

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 ...

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stem cells – Research | Lupus Foundation of America

Posted: August 22, 2016 at 8:46 am

Stem Cells and Lupus Research

The LFA believes expanding stem cell research will accelerate the pace of discovery on the potential therapeutic benefits of stem cells and help basic and clinical researchers learn how stem cells can be used to develop life-saving treatments.

Stem cells possess the potential to develop into many different types of cells in the body. They serve as a repair system for the body. There are two main types of stem cells: embryonic stem cells and adult stem cells.

Embryonic stem cells are taken from human embryos. They are found exclusively in early-stage embryos, from which all the bodys 200-plus types of tissue ultimately grow. They are the bodys master cells.

Adult stem cells are found in mature tissues that have already developed. They are more specialized than embryonic stem cells. The body uses these cells to replace other cells that die off throughout the normal course of life. As they are not from fetal tissue, adult stem cells do not have the same ethical concerns or restrictions that embryonic stem cells do. Current research in lupus focuses on adult stem cells, namely mesenchymal stem cells.

Mesenchymal stem cells (MSC) are derived from bone marrow, umbilical cords or other tissues and are anti-inflammatory. These anti-inflammatory cells have unique properties that make them attractive as therapy for autoimmune diseases. Unlike with other stem cells, MSCs lack the properties that enable the immune system to detect them as being foreign. Therefore:

MSCs have been studied in inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and scleroderma. In these studies, MSC treatment has been found to be effective and only minimal side effects reported.

Pioneering researchers from China have studied MSC treatment in over 200 people with lupus who had been unresponsive to standard lupus therapies. Preliminary results show that:

While the findings are groundbreaking, there was no control group in these early studies. This means that every participant received the stem cell treatment plus standard lupus therapies. A controlled trial is necessary to ensure that individuals who receive the MSC therapy plus standard lupus therapies do indeed respond better than closely matched participants who only receive standard therapy.

This first-of-its-kind phase II clinical trial of mesenchymal stem cells for the treatment of moderate to severe lupus could help lower medication dosage, diminish the long-term effects of lupus, stop damage to vital organs, and save lives. The trial is led by Drs. Gary Gilkeson and Diane Kamen from the Medical University of South Carolina and will be conducted at six research institutions around the country.

Stem cell therapy holds promise as a safe and effective alternative for people with lupus who do not benefit from the current treatments available. Adult stem cell research has provided hope to people with formerly incurable and devastating conditions, including Parkinsons disease, leukemia, heart diseases, multiple sclerosis, juvenile diabetes and osteoarthritis, as well as 80 other diseases.

Previous research using this type of therapy for lupus and other diseases reported minimal side effects. Like every potential new therapy, this treatment must be tested. We remain hopeful this procedure will prove successful and be included in the arsenal of treatments for lupus.

Only one drug is available that was developed specifically to treat lupus. It took more than 50 years for a new drug to be approved for lupus and it does not work for everyone. We need treatments for lupus, and this study provides hope for the future.

We are rallying support for this promising research so it will get the attention and research funding it deserves from public and private sources.

Previous research on adult stem cells has been promisingbut more testing is needed. Thats why we need your support. We are asking donors to consider supporting this effort with a special contribution. Our goal is to raise $500,000. The funding will enable researchers to treat initial participants. Ultimately, the study will be expanded through potential funding from the National Institutes of Health (NIH) and other sources.

If you'd like to support this research, consider making a dedicated gift.

To learn more about this study and determine if you are eligible to participate, visit the MSCs in SLE Trial page on ClinicalTrials.gov.

The LFA thus far has awarded 11 grants to advance basic and clinical adult stem cell research as a treatment for lupus. Learn more about all the investigators we have funded for stem cell research.

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The leukemic stem cell – PubMed Central (PMC)

Posted: August 22, 2016 at 8:44 am

Best Pract Res Clin Haematol. Author manuscript; available in PMC 2008 Mar 1.

Published in final edited form as:

PMCID: PMC1988840

NIHMSID: NIHMS28836

Craig T. Jordan, James P. Wilmot Cancer Center, University of Rochester School of Medicine, Rochester, NY;

Malignant stem cells have recently been described as the source of several types of human cancer. These unique cell types are typically rare and possess properties that are distinct from most other tumor cells. The properties of leukemic stem cells indicate that current chemotherapy drugs will not be effective. The use of current cytotoxic agents is not effective in leukemia because the agents target both the leukemic and normal stem cell populations. Consequently, new strategies are required that specifically and preferentially target the malignant stem cell population, while sparing normal stem cells. Several well known agents are lethal for the leukemic stem cell in preclinical testing. They include parthenolide, commonly known as feverfew, and TDZD-8. They have undergone various levels of preclinical development, but have not been used in patients as yet in the cancer setting. These drugs and combinations of existing therapies that target the leukemic stem cell population may provide a cure in this disease. This article summarizes recent findings in the leukemic stem cell field and discusses new directions for therapy.

Keywords: leukemic stem cell, parthenolide, TDZD-8

Approximately 50 years ago the concept was introduced that a stem cell may be connected to the origin and evolution of myeloid leukemia. To hypothesize the evolution of this disease, some form of normal stem or progenitor cell undergoes a mutation, giving rise to an entity that is functionally defined as a leukemic stem cell. The normal stem cells continue to differentiate into the hematopoietic lineage giving rise to erythrocytes, platelets, leukocytes, and granulocytes. The mutated stem cells have properties similar to the normal stem cells and can also differentiate into the hematopoietic lineage carrying the defect/s or can remain and accumulate as immature progenitor cells, also known as blast cells ().

Hypothesis of myeloid leukemia development

The chemotherapeutic agents used today effectively eradicate the blast cells in many patients. However, those same agents have very little if any activity at the level of the blast progenitor cell, the leukemic stem cell (LSC), which is biologically distinct from most of the cells that are found in a typical patient. An LSC is a functionally defined entity not necessarily named because it arises from a normal stem cell, but because it fulfills the same criteria used to define normal stem cells. These cells can undergo self renewal, are multipotent, and highly proliferative. The origin of the LSC has been the subject of considerable research in recent years.1

During normal developmental progression from stem cell to progenitor cell to mature cell, mutations may potentially occur at any point during this evolution, giving rise to a malignant entity. A mutation in a normal stem cell might give rise to a unit that could be considered an LSC. However, there is experimental evidence that suggests that mutations in a progenitor cell that no longer has all the characteristics of a stem cell can also give rise to an entity that can initiate and maintain leukemic disease.2 By extrapolation, it can be seen that more differentiated cells with the appropriate mutations may also give rise to the leukemic stem cell.

From a therapeutic perspective, the nature of the LSC may vary depending upon the stage during which it arose. Accordingly, drug resistance and various characteristics that are relevant to therapy may also differ, based on the origin of the diseased cell. Thus, it is possible to speculate that certain forms of leukemia that are relatively amenable to current therapies may derive from more differentiated cells that have certain intrinsic properties that are more readily addressed by conventional therapy. A plethora of new agents is available for treating leukemias, including kinase inhibitors, histone deacetylase inhibitors, cyclin D kinase inhibitors, heat shock protein inhibitors, methylation inhibitors, farnesyltransferase inhibitors, NF-kB inhibitors, and proteasome inhibitors.3,4 All of these agents affect specific mechanisms that may have gone awry. Despite the excitement and potential of all of these agents, there is not a single drug that has yet been validated as useful for eradicating the human LSC. An extremely important undertaking will be to fill this gap and to understand if and how all these new therapies are acting at the level of the stem cell.

Because anthracyclines, alkylating agents, nucleoside analogs, and topoisomerase inhibitors currently used in the treatment of acute myeloid leukemia (AML) often fail, they may not be targeting LSCs very effectively.5 In fact, there is no evidence that there is any selectivity or specific targeting of a leukemic versus normal stem cell by these agents. For example, Ara-C, which is a cycle-active agent commonly used in treating leukemia, shows virtually no activity with isolated LSCs.6 In contrast, this agent is effective when tested on blast cells from the same patient. There is a distinct difference in treating these two cell populations. When anthracyclines are tested in vitro, they are extremely cytotoxic both to LSCs and normal stem cells and there is no selectivity. However, there are ways that conventional therapies can be used in the appropriate combinations and appropriate methodologies, some of which have been previously published, to enhance the selective targeting and killing of leukemic stem cells.

LSCs can be isolated based on their cell surface markers using the currently available cell-sorting technologies. Once the leukemic stem cells have been isolated, they can be analyzed using the same techniques applied in any type of cancer cell to understand the specific mutations and pathways that propel growth and survival. Tumor-associated properties in the LSC could include mutations in the kinase domains, transcription factors, and tumor suppressors, or alterations in the growth and survival mechanisms mediated through NF-kappa B (NF-B) or PI3 kinase, or changes in physiology, glucose metabolism, or responses to oxidative stress, to name a few. The results obtained from these analyses of LSCs can be compared to the unique properties of normal stem cells, which are relatively rare, have a quiescent cell cycle status, can efflux drug, and have self-renewal properties. Based on these findings we will be able to effectively develop regimens that will target LSCs.2

From this relatively broad perspective, it is difficult to choose the most effective pathways to target. A great deal of knowledge has been amassed on agents that target mitogenic tyrosine kinase mutations, such as the BCR/ABL mutation by imatinib mesylate, which induces apoptosis of primary chronic myeloid leukemia (CML) cells.7 Another example is the Flt3 inhibitor CEP701, which induces apoptosis of primary AML blasts.8 However, there is no evidence that inhibiting these pathways is relevant to inhibiting the proliferating LSC.

As an alternative to inhibiting the activity of specific mutations in a cancer cell, it might be appropriate to consider the physiology of other unique mechanisms that maintain the viability and survival of these cells. Two such mechanisms have been suggested in the literature: the constitutive activation of the PI3 kinase pathway9 and NF-B,10 which are evident in LSCs. Inhibiting these two pathways might have therapeutic relevance. While no particular mutations or specific genetic events are associated with activation of the NF-B or PI3-kinase pathways, converging events, such as multiple different mutations, may feed into these pathways. However, such pathway modification may not be the only mechanism to produce leukemia.

Primitive human LSC populations can be selected by cell surface markers containing CD34+/CD38/CD123+ antigens. These cells are almost entirely quiescent, mimicking normal stem cells. As a result, cell cycle agents that are active in dividing cells will not be effective with this population. To characterize unique properties of LSCs, these purified populations have been examined using molecular analyses that look for activation of the NF-B pathway. As shown in , activation of the NF-B pathway can be readily detected. Normal hematopoietic stem cells do not show activation of NF-B. We believe that this is a leukemia-specific phenomenon.

NF-kB activity in primitive AML cells

Idarubicin with a proteasome inhibitor, parthenolide, and TDZD-8 are three examples of regimens that selectively target the leukemic stem cell. All three are able to inhibit NF-B activity. The combination of idarubicin with various classes of proteasome inhibitors mediates selective apoptosis in LSCs while sparing normal cells.10 The second agent, parthenolide, is a naturally occurring sesquiterpene lactone11 that also selectively kills LSC populations with minimal activity in normal stem cells. Continuing studies with this agent in colony-forming assays have been used to measure progenitor cell function. This pattern of selectivity continues with the third agent, TDZD-8, which is lethal only to the leukemic population. Data on TDZD-8 is yet unpublished. From a preclinical standpoint, these types of molecules appear promising.

Parthenolide is a small molecule that is the active compound in the plant known as feverfew, which has been used for centuries in herbal remedies to treat headache and inflammation. It is a potent inhibitor of NF-B. Parthenolide selectively ablates myeloid leukemia cells at an IC50 of 2.5 M and causes apoptosis in primary human AML cells and blast crisis CML (bcCML) cells. AML progenitor and stem cells were analyzed in in vitro colony forming assays in a nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft mouse model. Parthenolide preferentially targeted both AML progenitor and LSC populations.11 The only drawback is that parthenolide is not a good candidate for pharmacologic development because it is not particularly water soluble. We have collaborated with Peter A. Crooks, PhD, at the University of Kentucky to develop analogs of parthenolide. The best candidate so far is a fumarate salt of dimethylaminoparthenolide. Its solubility in water is ~1000-fold greater than parthenolide and maintains its LSC-specific activity. It has been picked up by the NCIs RAID program and is currently undergoing further preclinical development. We hope to see this agent entering trials relatively soon.

TDZD-8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) belongs to a family of molecules with an interesting history. A research group from the University of Madrid tested agents to inhibit the enzymatic activity of glycogen synthase kinase-3 beta (GSK-3 with the aim of treating neurologic diseases, such as Alzheimers, in which the enzyme GSK-3 is very active. The agents they tested also have NF-B inhibitory activity. One of these compounds, TDZD-8, is a non-ATP competitive inhibitor of GSK-3 (IC50=2M), binds to the active site of GSK-3, and inhibits the activities of Cdk1/cyclin B, casein kinase-II, and protein kinase A and C (IC50>100M). Preliminary data show that TDZD-8, similar to parthenolide, selectively induces the death of primary AML progenitor cells.

Further preliminary studies compared the efficacy of treating the AML progenitor cells with either parthenolide or TDZD-8. An overnight culture of AML progenitor cells expressing CD34+CD38- treated with either parthenolide or TDZD-8 showed that survival is rapidly impaired by TDZD-8 in comparison to parthenolide (half life of 120 minutes for cells in the presence of TDZD-8 vs > 360 minutes for those in the presence of parthenolide), relative to untreated controls. By contrast, there was very little to no activity in normal patient specimens.

In terms of the kinetics of these two agents, cells were treated with TDZD-8 or parthenolide at intervals of 30, 60, 120 1440 minutes and then assayed for viability. It became readily apparent between 6 and 12 hours that cells were dying in the presence of parthenolide. With TDZD-8, these cells were killed in as little as 30 minutes. When AML progenitor cells were introduced into NOD/SCID mice to rigorously assess stem cell potential, TDZD-8 inhibited the engraftment of AML leukemic stem cells, but did not significantly inhibit engraftment of normal hematopoietic stem cells. In a phenotypic analysis, cells were taken from the cell viability assay and introduced at each of the same time points into a functional assay such as progenitor cell colony-forming assays with striking results. The cells from the previous assay that were transferred into a functional assay had no detectable colony forming activity in as little as 30 minutes in the presence of TDZD-8. By comparison, parthenolide required an overnight time lapse before readily evident cell kill was observable. The mechanism of TDZD-8 is being investigated; however, from an empirical perspective, the extremely fast cell death with this agent is a very exciting development.

The two different agents discussed in this paper, parthenolide and TDZD-8, are chemically distinct entities. These compounds have the property of mediating cell death and inhibiting leukemic stem cell-specific activity. The empirical observation is that there are two types of stimuli required to bring about leukemia-specific cell death. The first is the inhibition of a survival pathway. The NF-kappa B and PI3 kinase pathways are affected by these drugs, but there could be others. Yet inhibiting pathways alone is not particularly toxic. However, when combined with a stimulus in the form of oxidative and/or genotoxic stress, the two signals together appear to be highly selective in killing leukemic stem cells, but are not particularly toxic at the normal stem cell level. Agents like parthenolide and TDZD-8 appear to simultaneously deliver both signals (). A general criterion for selecting therapeutic regimens could be the ability of the agent to deliver both signals simultaneously.

Hypothesis to propel selective death in leukemic stem cells

In developing regimens that are more selective to leukemic stem cells, these agents should be developed for patient use and their activity should be validated at the level of stem cells. Similarly, when these regimens are used in patients, patient outcomes should be assessed in terms of the relative efficacy of targeting leukemic stem cells in vivo. The assays are currently available and can be applied to determine this.

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Skeletal muscle stem cells – National Center for …

Posted: August 22, 2016 at 8:43 am

Reprod Biol Endocrinol. 2003; 1: 101.

1Department of Molecular and Cell Biology, Center for Regenerative Biology, University of Connecticut, 1392 Storrs Road Unit 4243, Storrs, CT 06269-4243, USA

Received 2003 Aug 20; Accepted 2003 Nov 13.

Satellite cells are myogenic stem cells responsible for the post-natal growth, repair and maintenance of skeletal muscle. This review focuses on the basic biology of the satellite cell with emphasis on its role in muscle repair and parallels between embryonic myogenesis and muscle regeneration. Recent advances have altered the long-standing view of the satellite cell as a committed myogenic stem cell derived directly from the fetal myoblast. The experimental basis for this evolving perspective will be highlighted as will the relationship between the satellite cell and other newly discovered muscle stem cell populations. Finally, advances and prospects for cell-based therapies for muscular dystrophies will be addressed.

Skeletal muscle is subject to constant injury resulting from weight bearing, exercise, and trauma, thereby requiring an ever-available, renewable source of cells for muscle repair and regeneration. Since its identification, the satellite cell has been a popular candidate for the adult skeletal muscle "stem cell" [1]. Residing dormant beneath the basal lamina of mature skeletal muscle fibers, this cell is ideally located for timely repair of degenerating muscle fibers. Additionally, these quiescent cells are activated to proliferate upon muscle injury, a necessary step towards generating sufficient numbers of myoblasts for muscle differentiation and myotube formation. However, the identification of multiple stem cell populations resident in skeletal muscle has added further complexity to understanding the process of muscle regeneration. In this mini-review, we will briefly examine the molecular and morphological characteristics of the satellite cell, its role in muscle regeneration, and discuss outstanding questions regarding its origin, developmental potential, and uses in myoblast therapy.

Although the developmental origin of satellite cells remains unknown, in vertebrates, the majority of skeletal muscle progenitors arise in the somites. Somites are transient epithelial spheres that pinch off of the paraxial mesoderm lining both sides of the neural tube. Myogenic precursors are first identified in the dermomyotome, an epithelial layer located in the dorsal compartment of the somite. These precursors are characterized by their expression of the paired box transcription factors Pax-3 and Pax-7; in response to signals such as Wnts and Sonic hedgehog from surrounding embryonic structures, the myogenic determination genes Myf-5 and MyoD are activated [3]. Coinciding with the down-regulation of Pax gene expression, muscle precursor cells committed to the skeletal muscle lineage (myoblasts) translocate to the subjacent myotome, where the muscle regulatory factors Myogenin and MRF4 direct differentiation and fusion into multi-nucleated myofibers.Satellite cells are first apparent towards the end of embryogenesis, and function as a primary source for the myogenic cells required for post-natal muscle growth [2].

In adult muscles, dormant, Pax-7-expressing satellite cells reside between the plasmalemma and basal lamina at frequencies that vary with age, muscle fiber type, and species [4]. The activation of satellite cells in vivo can be induced by muscle fiber injury brought on by acute injury [5-7], exercise [8-10], and denervation [11]. Upon injury, satellite cells are stimulated to re-enter the cell cycle to generate a pool of proliferating myogenic precursors analogous to the embryonic myoblasts, while the inflammatory response mounted by the immune system clears affected myofibers [2]. Recently, certain Wnt-family members were found to be up-regulated in muscle following injury, suggesting a parallel to myogenic signaling pathways in the embryo [12]. Additionally, up-regulation of Myf-5 and MyoD occurs at the injury site in proliferating satellite cells indicating cell commitment [13-17]. Pax-7 expression declines with the up-regulation of MRF-4 and Myogenin, and differentiated myocytes fuse to new and existing fibers as part of the repair process. One of the hallmarks of regenerating myofibers is the centrally located position of the myonuclei; upon maturing, muscle fiber nuclei are located along the cell periphery [4]. Notably, repeated cycles of injury and regeneration do not appear to deplete satellite cell numbers, suggesting that these cells have the ability to self-renew [2].

Satellite cells were initially identified in frog leg muscles by electron microscopy [1], and subsequently have been identified in all higher vertebrates. In humans and mice, these quiescent [18], non-fibrillar, mononuclear cells are most plentiful at birth (estimated at 32% of sublaminar nuclei) [19]. The frequency declines post-natally, stabilizing to between 1 to 5% of skeletal muscle nuclei in adult mice [2]. Satellite cell frequency varies in different muscles, likely as a function of variation in fiber type composition (i.e. slow oxidative, fast oxidative, or fast glycolytic fibers). For example, the mouse soleus muscle, which is predominantly made up of slow oxidative fibers, has a higher number of satellite cells than the extensor digitorum longus (EDL) muscle, which primarily contains fast glycolytic fibers. Additionally, the absolute numbers of satellite cells increases in the soleus but not the EDL between 1 and 12 months of age, although the proportion of satellite cells decreases in both muscle types with increasing age [20]. In humans, the proportion of satellite cells in skeletal muscles also decreases with age, which may explain the decreased efficiency of muscle regeneration in older subjects [21]. Satellite cells from aged muscle also display reduced proliferative and fusion capacity, as well as a tendency to accumulate fat, all of which likely contribute to deteriorating regeneration capability [22,23]. That endurance training can offset the decline in satellite cell number with age suggests that poorer regeneration is not simply a result of limited replicative potential of older satellite cells [24].

Several signals and growth factors have been implicated in promotion of satellite cell activation and proliferation (Figure ). For example, the Notch signaling pathway, which is activated upon muscle injury, regulates satellite cell transition from quiescence to proliferation in single fiber cultures, thereby expanding the myoblast population in injured muscle [25]. Basic fibroblast growth factor (bFGF) stimulates satellite cell proliferation while inhibiting differentiation [2]. bFGF also promotes muscle regeneration in mdx mice [26], which undergo repeated cycles of degeneration and regeneration resulting from a mutation in the dystrophin gene; in humans, deficiency of dystrophin causes Duchenne muscular dystrophy [27,28]. In addition to expressing all known FGF receptors [29,30], satellite cells also express the tyrosine kinase receptor c-met [16,31]. The c-met ligand, hepatocyte growth factor/scatter factor (HGF/SF), is also a known activator of satellite cells [29,32].

Model for the development, activation, and maintenance of the satellite cell. Upon skeletal muscle injury, quiescent satellite cells expressing Pax-7 and Foxk1 are activated to proliferate, up-regulating the myogenic determination factors, MyoD and Myf-5 ...

Targeted deletion of the gene encoding the Forkhead/winged helix transcription factor Foxk1 [previously known as myocyte nuclear factor (MNF)], which is expressed in quiescent satellite cells, causes a severely runted phenotype, and cardiotoxin-induced muscle regeneration is delayed and accompanied by prominent accumulation of adipose cells, suggesting a defect in skeletal muscle commitment [33]. Interestingly, the myopathy associated with the Foxk1 mutant is rescued when bred into a p21-null background. p21 is up-regulated in Foxk1-null muscles, and while mice lacking this cyclin-dependent kinase inhibitor show a defect in satellite cell differentiation, double mutants exhibit normal muscle growth and regeneration, suggesting that p21 is a downstream target of Foxk1 [34,35].

The muscle determination gene MyoD is also required for normal muscle regeneration [36]. Regenerating muscles in MyoD-null animals accumulate high numbers of mononuclear cells and have few differentiated myotubes; this phenotype is exacerbated in an mdx background, with MyoD-/-; mdx muscles exhibiting severely reduced cross-sectional area and mass. MyoD-null animals exhibit increased numbers of satellite cells, suggesting that the cells fail to progress through the differentiation program and instead participate in self-renewal [36]. The abnormal proliferation observed with MyoD-null adult myoblasts and failure to up-regulate the muscle differentiation factors MRF-4 or Myogenin under differentiation conditions support this hypothesis [37,38]. In addition, MyoD-null satellite cells express increased levels of Myf-5 [37,38]. In embryos lacking MyoD, myogenesis is dependent on Myf-5 and vice versa: while single mutant embryos have normal muscles at birth, MyoD-/-; Myf-5-/- double mutant embryos fail to develop myoblasts or myotubes [39-41]. Given the defects in muscle regeneration observed in adult MyoD mutants, it is evident that the functional redundancy between MyoD and Myf-5 that ultimately rescues embryonic muscle development is not sufficient to rescue myogenesis in injured muscle.

Interestingly, while traditionally thought to be committed to the skeletal muscle fate, it is now evident that muscle stem cells, including satellite cells, are multipotent. For example, bone morphogenetic protein (BMP) treatment activates osteogenic markers while down-regulating MyoD in C2C12 myoblasts, an immortalized cell line derived from mouse limb muscle [42,43]. Additionally, treatment with thiazolidinediones and fatty acids converts C2C12 cells to the adipogenic cell fate [44]. Primary myoblast cultures from adult muscles respond similarly to C2C12 cells in the presence of strong osteogenic and adipogenic inducers; interestingly, satellite cells derived from intact single fiber cultures (and thought to be more representative of true myogenic stem cells) spontaneously form adipocytes and osteocytes when cultured on Matrigel, a soluble basement membrane matrix lacking strong osteogenic or adipogenic signals [45]. The finding that undifferentiated cells in adult myoblast cultures co-express MyoD, Runx2, and PPAR, key regulators for myogenesis, osteogenesis, and adipogenesis, respectively, supports the hypothesis that satellite cells have a multipotential predisposition [46].

The plasticity of muscle stem cells has also been demonstrated using ex vivo approaches. Muscle stem cells isolated via serial preplating enrich for a population of cells which, in addition to contributing to regenerating myofibers when injected directly into dystrophic muscle, are detected in differentiated vascular and nerve cells [47,48]. Furthermore, these cells, which express the myoblast markers desmin and MyoD, are sufficient to completely heal skull defects in vivo when engineered to express BMP [49]. These muscle-derived stem cells are also capable of reconstituting bone marrow in lethally irradiated mice [50].

Another muscle-based stem cell with hematopoietic potential is the muscle side population (SP) cell, which can be isolated based on its specific exclusion of the vital dye Hoechst 33342 [51]. Initially sorted from bone marrow derived (BMD) stem cells by FACS analysis and observed to possess the majority of hematopoietic stem cell activity in bone marrow [52], SP cells have since been identified in a variety of tissues, including skeletal muscle, brain, heart, spleen, kidney and lung, although they are notably absent in peripheral blood [53]. It is important to note that the relationship between these different SP populations, and whether or not they derive from a common precursor, remains to be determined. Muscle SP cells reconstitute bone marrow in lethally irradiated mdx mice, although less efficiently than BMD SP cells. Interestingly, donor-derived nuclei also appear in regenerating muscle fibers after bone marrow reconstitution, indicating a contribution by the hematopoietic system in muscle repair [51,54]. The heterogeneity of muscle stem cells is underscored by the observation that SP cells within normal, uninjured skeletal muscle can be distinguished as positive for the hematopoietic marker CD45 (and poorly myogenic) or CD45-negative (a population that readily differentiates along the myogenic pathway) [55]. The CD45-positive subpopulation of cells has also been shown to contribute to neo-vascularization in regenerating muscle, whereas the CD45-negative population does not [56]. Interestingly, Wnts 5a, 5b, 7a and 7b, which are up-regulated in myoblasts and myofibers of regenerating muscle, convert the normally resistant CD45-positive muscle SP fraction to the myogenic program; this property to induce a switch in fate could contribute to the recruitment of much-needed progenitors upon injury [12].

A recent study of the Pax-7-null mouse revealed that this paired box transcription factor is essential for satellite cell formation. In addition to exhibiting severe muscle deficiency at birth and premature lethality, Pax-7 mutants are completely devoid of satellite cells [57]. However, while this observation demonstrates the requirement for Pax-7 in satellite cell formation, it remains to be seen whether the satellite cell arises from a pre-determined myoblast in the dermomyotome, a fetal myoblast, or from a non-somitic progenitor. Satellite cells may originate from specified Pax-7-positive cells prior to the activation of Myf-5 and MyoD, and thus represent a true precursor to the myogenic lineage. Alternatively, satellite cells may arise from determined myoblasts which, instead of differentiating, continue to proliferate until withdrawing from the cell cycle and taking up residence beneath the basal lamina of myofibers. While relatively little is known about the cis regulation of the Pax-7 gene, the extensive characterization of Myf-5 and MyoD regulatory elements [3,58] can be used to determine if satellite cells originate from a Myf-5 or MyoD-positive population by in vivo cell tracing. Interestingly, while Pax-7-null animals lack satellite cells, the muscle SP population remains intact, although exhibiting increased hematopoietic potential; Pax-7 may direct specification of pluripotent SP cells to satellite cells [57,59].

The observation that various non-muscle stem cells can participate in skeletal muscle regeneration has expanded the candidate pool for the satellite cell precursor. For example, myogenic potential has been demonstrated in vivo by mesoangioblasts, which are vessel-associated stem cells [60-62], neural stem cells [63], and, as mentioned previously, bone marrow cells [64,65].

Bone marrow cells have long been known to have myogenic potential [66,67]. Direct injection of -galactosidase-positive bone marrow cells into cardiotoxin-injured muscle gives rise to labeled myofibers, although at a lower frequency than injected satellite cells [64]. Interestingly, bone marrow cells contribute directly to regenerating myofibers in lethally irradiated mdx bone marrow transplant recipients [68]. Surprisingly, in the absence of myogenic induction, a subset of bone marrow cells in mdx mice are positive for both early and late myogenic markers including Pax3, MyoD, and myosin heavy chain, suggesting that muscle commitment and differentiation are underway [69]. Also intriguing is the finding that GFP-labeled BMD cells take up residence beneath the basal lamina of skeletal muscle fibers in irradiated transplant recipients following injury (in this case, an exercise model), with subsequent injury provoking an increased contribution of BMD cells to regenerated muscle fibers [65]. This suggests that satellite cells are maintained in regenerating fibers through self-renewal as well as replenishment from the bone marrow. It remains to be seen what proportion of satellite cells arise anew with each round of injury, and whether multiple rounds of injury results in a complete turnover of host satellite cells with donor bone marrow cells.

The use of muscle stem cells for therapeutic purposes holds much promise for treatment of diseases affecting skeletal muscle, including muscular dystrophy [70]. Dystrophic muscles that receive myoblast transplants exhibit some dystrophin-positive myofibers, and persistence of donor fibers in regenerated muscles is observed [71-73]. However, certain roadblocks hinder the efficacy of this therapy, including the limited migration of donor cells into dystrophic muscle and problems with poor donor cell survival and inefficient myogenic contribution. Advances have been made in identifying chemotactic factors and cell surface molecules that enhance the migration of transplanted cells [74-76]. In addition, careful selection of donor cells has been shown to enhance efficiency of rescue and cell survival in transplant hosts. In particular, Huard and colleagues have found that their serial preplated muscle stem cell cultures display enhanced proliferative capabilities and readily contribute to regenerating muscle while failing to trigger a strong immune response [47,77]. Furthermore, CD45-positive muscle SP cells also contribute to regenerating muscle with high efficiency [54,59].

The use of bone marrow transplants for treating muscular dystrophy has been contemplated as an alternative therapy to myoblast injection and, as mentioned previously, BMD cells do contribute to regenerating muscle. In fact, bone marrow derived nuclei have been identified in muscle biopsies from a 15-year-old patient who received a bone marrow transplant at age 1, and was diagnosed with a mild case of Duchenne muscular dystrophy at age 12 [78]. While this demonstrates the longevity of transplanted cells in muscle, it remains to be seen whether the contribution of these cells to regenerating muscle is responsible for the mild form of the patient's disease. Intriguingly, intra-arterial injection of wild-type mesoangioblasts into mice suffering from limb girdle muscle dystrophy results in complete functional recovery of all affected muscles [62]. This presents a promising solution to difficulties encountered with myoblast transplantation therapy, and makes all muscles accessible for treatment. This is especially important for the treatment of essential muscles such as the diaphragm, impairment of which results in severe respiratory problems.

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Posted: August 21, 2016 at 6:50 pm

Cato Research (CATO) is an international regulatory and clinical contract research organization (CRO) that has been delivering successful outcomes for its clients for more than 27 years. Through highly qualified and experienced personnel located in offices across North America, Europe, Israel, and South Africa, supported by strategic alliances with selected regional CROs, CATO offers tried-and-trusted international coverage.

CATO has the advantage of integrated regulatory, clinical, and scientific expertise. Our highly talented and experienced international team offers services from preclinical through IND (or equivalent), clinical development, and marketing approval to Phase 4 postmarketing research. Our track record includes successes across many different health products, including drugs, biologics, vaccines, cell therapies (including stem cells), nutriceuticals, medical foods, devices, and diagnostics. In terms of diseases and indications, in more than 27 years of operation, CATO has been involved in a wide variety of projects with a dominant focus in oncology, neurology/CNS, and orphan drug indications. This experience includes leadership of, and involvement in some of the most challenging and innovative products.

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Stem Cells | USC News

Posted: August 20, 2016 at 1:43 am

Health 2 Zika proteins responsible for microcephaly identified

Its the first study to examine Zika infection in human neural stem cells from second-trimester fetuses, USC researchers say.

Andy McMahon and his colleagues investigate ways to help the millions who suffer from the chronic ailment.

The condition is more widespread in the animal kingdom than scientists suspected, USC study finds.

NIH Pathway to Independence Award will help Lindsey Barske transition to the faculty stage as she hopes to learn more about human birth defects.

Researchers examine the stimulation of the inner ears sensory cells, which could ultimately help the deaf.

USC researchers hope to usher in new treatments for patients with muscular dystrophy.

The scientists receive the first Broad Innovation Awards for their critical analysis of Lou Gehrigs disease and immune systems.

Findings could impact development of clinical strategies to treat cancers of the lung, breast and prostate.

The objective of one current research proposal is to push the frontiers of stem cell and tissue engineering technologies.

Altering transplantation dose could improve outcomes for patients and ultimately save lives.

USC Stem Cell researchers show that cisplatin causes more acute hearing loss in mice with the equivalent of Cockayne syndrome.

Scientists hope that lessons learned from zebrafish jawbone regeneration might hold promise for healing severe human bone fractures.

Researchers discover that two types of molecular signals work to control where and when stem cells turn into facial cartilage.

Colleagues explain how a shared gene directs the development of bone-forming cells.

Influenced by the Jesuits, a USC researcher believes in service, which involves an expansion of knowledge through research and education.

Tracy Grikscheit awarded $7.1 million grant by the California Institute of Regenerative Medicine.

A human has nearly 100 times more nephrons, the functional units of the kidneys, than a mouse.

Next-generation scientists at USC Stem Cell mini-symposium discuss research of anemia and cancer.

Gabriel Linares seeks therapies for patients with Lou Gehrigs disease.

USC-affiliated faculty members aim to cure diseases using stem cells as tools.

Doctoral student in USC Stem Cell lab helps to identify roles for a family of genes.

Prkci influences whether stem cells self-renew or differentiate into more specialized cell types.

The new one-stop shop allows scientists to take stem cell research to the next level and eventually develop translational therapies.

Albert Kim applies his expertise to the challenge of kidney regeneration.

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Cell Therapy – Celgene

Posted: August 20, 2016 at 1:42 am

Celgene Cellular Therapeutics (CCT) is at the forefront of cell therapy research and development. Our lead cell therapy candidates are derived from healthy, full-term placenta, a remarkable organ shared by mother and baby. From this immunologically privileged source, we have generated novel cell therapy candidates with immunomodulatory, anti-inflammatory, angiogenic and reparative properties.

CCTs placenta-derived adherent cells (PDAC cells) have been extensively characterized (see our Publications) and display multiple activities including the ability to stimulate natural repair processes, with the potential to transform the treatment of a broad range of serious debilitating diseases including autoimmune, vascular and neurodegenerative disorders. Specific formulations of PDAC cells are our lead clinical candidates PDA-001 (intravenous) and PDA-002 (intramuscular), currently in clinical trials for Crohns disease, Peripheral Artery Disease with Diabetic Foot Ulcer and other indications.

Cell therapy research teams focus on the discovery and early development of novel cell-based therapeutics. We have developed a portfolio of unique clinical candidates with broad therapeutic potential, including human placenta-derived stem cells (HPDSC), natural killer (NK) cells and amnion-derived adherent cells (AMDAC cells). We are industry leaders in adult stem cell isolation, cell culture, characterization, functional interpretation and translational medicine. Our state-of-the-art in vitro, preclinical and translational biology approaches conducted in-house and in collaboration with international expert groups, are specifically designed to elucidate the complex mechanisms that are associated with live cell-based therapeutics

We are constantly searching for new ways to bring innovative solutions to unmet medical needs. Celgene is a leader in cancer treatments, and we are investigating novel cell therapies to expand this arsenal, both in-house and through alliances with other leaders. Our collaborations with bluebird bio and Baylor College of Medicine on chimeric antigen receptor-modified T-cell-based (CAR T) therapies genetically modifying a patients own cells to fight cancer have the potential to revolutionize patient care in a range of hematological and solid malignancies.

CCT is proud to work with federal research agencies and has been a performer for the Defense Advanced Research Project Agency (DARPA) since 2008. CCT has led two international consortia to develop advanced stem cell manufacturing and differentiation methods and hematopoietic stem cell-based therapeutic concepts and product candidates, leading to breakthrough findings in these areas.

CCT is a world leader in cell therapy process development and clinical manufacturing. We possess in-house GMP facilities and development laboratories for processing allogeneic and autologous cells from a variety of cell sources while maintaining the highest level of regulatory compliance. We have broad experience in developing, optimizing, scaling up and validating cell therapy processes and assays, from donor tissue procurement through product supply and distribution. Our integrated Technical Operations laboratories and staff share the same location, enabling us to gain process understanding from production operations and to translate development activities into manufacturing science. In summary, CCT Technical Operations has all systems, personnel, facilities, and capabilities necessary for taking a cell therapy product from discovery into the clinic and to commercialization.

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Cell Therapy - Celgene

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Balance Cell Therapy System| 12oz for Normal Hair

Posted: August 20, 2016 at 1:42 am

Recommended For You

Balance Cell Therapy System

12oz

$96.49 $111.85 Value! New

Normal Hair Types seeking a balance of moisture and volume.

The Balance System contains the Volume Therapy Shampoo to gently cleanse, Cell Therapy to treat and the Moisturizing Conditioner to hydrate strands for thicker, stronger, longer hair.

The Balance System consists of three products to cleanse, treat, and moisturize.

Apply Ovation Volume Therapy Shampoo to wet hair. Gently work into lather at the scalp and lightly massage through ends, then rinse. Follow with Ovation Cell Therapy Hair Treatment.

Cell Therapy Routine Treatment: Use 2-3 times per week. Prepare hair by washing with an Ovation Shampoo. Massage Ovation Cell Therapy into scalp and comb through. Leave on for 3-5 minutes and rinse thoroughly.

Apply Ovation Moisturizing Conditioner following Ovation Volume Therapy Shampoo or Ovation Cell Therapy Hair & Scalp Treatment. Gently massage through hair. Rinse thoroughly.

Style as desired.

I can see a remarkable improvement in the thickness, strength and length of my hair....Even my stylist has made these comments as well!

Cynthia G.

I love Ovation Cell Therapy. I can tell an instant difference from even one wash. I tell everyone I know about it.

Samantha F.

I bought Cell Therapy for my daughter and her hair is looking beautiful. Her hair was damaged and Cell Therapy has been a lifesaver. She is on her way to hair recovery.

Angela H.

I love Cell Therapy...made a world of difference for my hair and for my confidence!!

Rachel H.

I've experienced far less shedding and breakage since I've started using Cell Therapy. Thank you! My mother loves it too!

Ashley F.

What ingredients in Cell Therapy Hair & Scalp Treatment make it effective?

Cell Therapy Hair Treatment nourishes the hair and scalp with proteins, vitamins, and amino acids that bind and absorb into the hair shaft to create thicker, longer, stronger, and visibly healthier hair.

How long will a 12oz System typically last?

A 12oz Cell Therapy System lasts around 3 months based on average use.

How often should I use Cell Therapy Hair & Scalp Treatment?

We recommend incorporating Cell Therapy into your haircare washing regime 2-3 times per week. Do not exceed three Cell Therapy Hair Treatment applications between shampoos. Overuse of Cell Therapy Hair Treatment may result in stiff or weighed down hair, and possible breakage. Call us for a personalized regime to work Cell Therapy into your haircare routine at 888.411.3252 Option 2.

Should I use Cell Therapy Hair & Scalp Treatment if I have a Brazilian Blowout or Keratin Treatment?

We recommend consulting with your stylist before incorporating Cell Therapy into your hair care routine if you have a Brazilian Blowout or Keratin Treatment. Cell Therapy contains keratin and proteins which could cause protein overexposure, leading to dry, brittle hair. Please keep in mind our sulfate-free shampoos and conditioners are safe to use anytime.

Can I use Cell Therapy Hair & Scalp Treatment with other hair growth or strengthening products?

Ovation products are not medicated; we do not recommend that you use in combination with medicated hair growth products. Please consult your stylist or dermatologist before incorporating any other product into our Ovation Hair Care Systems.

How much Cell Therapy Hair Treatment should I apply with each treatment?

A quarter to half dollar size amount, depending on hair length.

Which Cell Therapy System should I use?

Our Cell Therapy Systems are the perfect way to experience the amazing benefits of Ovation products. These three-step systems provide a Shampoo, Cell Therapy and a Conditioner that work synergistically to maximize performance and cost-savings. The Cell Therapy formula is the same in each system. The difference between the systems resides in the moisture levels in the Shampoo & Conditioner which complete the system. We recommend choosing your Cell Therapy System based on your hair type and/or hair care concern. If you would like a personalized recommendation, our Personal Account Representatives are available to help Monday through Friday 7:30 a.m. to 5:00 p.m. Pacific at 888.411.3252 Option 2.

How long do Ovation products take to work?

Our product formulations are hair maximizers, and most customers notice that their hair appears stronger and thicker after a few uses; however, typical results are apparent after 4-6 weeks. Results can vary for each client.

Can I use Volume Therapy Shampoo every day?

Yes. Our Volume Therapy Shampoo is a gentle cleanser that is safe and effective for every day use.

Is the Volume Therapy Shampoo paraben and sulfate free?

Yes, the Volume Therapy Shampoo provides a gentle cleanse.

Is the Moisturizing Conditioner a leave-in treatment?

Our Moisturizing Conditioner is not a leave-in treatment. For deeper hydration, we recommend adding the Intensive Repair Hair Mask to your hair care regimen.

Are your products gluten free?

Yes.

Are your products Paraben free?

Yes.

Do you have a fragrance-free line?

Not at this time.

Do you perform animal testing?

No.

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Balance Cell Therapy System| 12oz for Normal Hair

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Biotechnology Teacher Resources Online | North Carolina …

Posted: August 19, 2016 at 2:48 am

Animal Biotechnology: The Movie

Animal Biotechnology and Genomics is a half-hour movie on various topics related to animal biotechnology. Accompanying fact sheets are provided.

APSnet: K-12 Plant Pathways to Science is intended to assist teachers develop interactive laboratories and demonstrations that teach the basic principles of science and scientific method. The site includes an Illustrated Glossary of Plant Pathology with pronunciation guides.

The Bioinformatics in Motion Project is a library of interactive animations for teaching high-level bioinformatics algorithms and application.

BioInteractiveis an interactive database of free resources for educators from HHMI. Search by topic and type of material to find animations, videos, classroom resource materials and more on many different health and basic science topics.

The National Science Digital Library is the nation's online portal for education and research on learning in Science, Technology, Engineering, and Mathematics with resources for K-12 students.

This section of theCBC Digital Archives includes archived clips of radio and television programs on biotechnology topics.

Discovery Education, the Institute of Food Technologists (IFT), and the IFT Foundation have partnered to develop this unique program, designed to introduce high school students, teachers, counselors, and parents to the remarkable world of food science and technology, and the exciting career opportunities in the field.

DNA From the Beginning is an "animated primer that covers 75 experiments that made modern genetics. Each chapter is self-contained, and they cover a wide range of topics related to genetics. Each of the 75 experiments explains the background, provides animations, galleries, videos and interactive elements.

DNA Interactive contains multimedia presentations on DNA, gene manipulation, the human genome, applications and the history of genetics. The DNAi Teacher Guide provides 15 downloadable lesson plans.

The DNA Television Series and accompanying web episodes and lesson plans are a valuable resource for teachers and students about the general history and applications of DNA.

The DNA Learning Center provides links to websites that focus on both genes and DNA. From the beginning of DNA, to the history of corn, to the insides of cancer, each site contains animated pictures that help students grasp the concepts.

Learn.Genetics provides educational materials on genetics, bioscience and health topics.

GetBiotechSmart contains videos and lesson plans for teachers around topics of plant biotechnology. The site also provides information about the Excellence in Biotechnology Education Grant, sponsored by the United Soybean Board.

Greenomes provides laboratory and bioinformatics exercises to introduce students to modern plant genetics and genome research.

The Harvest of Fear website is designed to accompany the PBS Nova program "Harvest of Fear." It contains a variety of stand-alone information and interactive activies to learn about genetically modified food.

Heal, Feed, Sustain is a 24-minute video that follows three students as they travel to three North Carolina companies to learn about biotechnology. It also features a free teacher guide with seven student activities, activity extensions, a curriculum connection matrix and more background information about biotechnology.

K8 Science is a multimedia resource that includes information , lesson plans and slide sets on scientific topics related to current events.

The Marian Koshland Science Museum of the National Academy of Sciences provides a multimedia tour of various museum exhibits with links to activities and lessons that describe applications of DNA technology.

Graphics teach what a microarray is and how these tools can be employed to simultaneously measure changes in gene expression across the genome of an organism.Students can conduct two different virtual experiments, one measuring changes in gene expression in yeast, and another which measures the effect of a bronchoconstrictor drug on gene expression in asthmatic and non-asthmatic mice.Along the way students learn about experimental design, how microarrays measure changes in gene expression, and methods of data analysis.This resource is geared primarily toward advanced high school and college students.

All of the lessons in this video library have been contributed by BLOSSOMS partners from around the world, including educators from MIT and institutions in Jordan, Pakistan, and other partnering countries. Each lesson includes Teacher Guides and other resource materials, and the site is searchable by education standards.

Molecular Movies presents an organized directory of cell and molecular animations.

Nanomedicine Explorer is a virtual exhibit dedicated to cancer nanomedicine.

NCLT provides a portal to educational resources that are meant to serve students, teachers, faculty, researchers, parents and professionals who are engaged in the learning, teaching, research & development of nanotechnology. NCLT is an NSF-sponsored program.

The Teacher Center provides a wide variety of information and resources for teachers, including lesson plans, state by state facts, and much more. National Agriculture in the Classroom isalso the publisher of an E-zine for educators called AgroWorld.

Over 400 peer-reviewed case studies are provided in a searchable database for use in the science classroom. A majority are written by science faculty from the U.S. and Canada, and teaching notes are provided.

Lab TV provides two seasons of webisodes that demonstrate research coming out of Department of Defense labs. Also, from the same website, click on Personal Stories to hear about careers in science and engineering.

The National Nanotechnology Infrastructure Network (NNIN) provides resources, programs, and materials to enhance an individuals knowledge of nanoscale science and engineering and its application to real-world issues. The network is made up of nanotechnology facilities at universities around the country.

The NIGMS provides e-publications, interactive online quizzes and puzzles, posters, fact sheets and curriculum supplements from this Science Education website.

Nova science Now provides text, video, and multimedia content from the PBS Nova TV series.

The UK-based Nuffield Council on Bioethics provides an Education page with teaching resources on five main topics: biofuels, personalized healthcare, dementia, forensics, and research animals.

PBS LearningMedia is a digital media service for educational use from public broadcasting and its partners. It provides free, instant access to the best of public media , including videos, interactives, audio, photos, and in-depth lesson plans.

PlantingScience is a learning and research resource, bringing together students, plant scientists, and teachers from across the nation. Students engage in hands-on plant investigations, working with peers and scientist mentors to build collaborations and to improve their understanding of science. A few open lesson plans are available in addition to the mentoring resources.

Science 360 is a site dedicated to scientific information. The Biotechnology Series is made up of three educational programs. The program topics covered are designer foods, genetic engineering, and stem cells demystified. Each program contains a video, lesson links, resources for teachers and students, and key vocabulary lists.

ScienceDaily Biotechnology features a collection of videos meant to supplement text articles written for an adult audience.

HHMI provides a list of short video interviews with scientists about their various career paths. There are more than 70 videos available "On Becoming a Scientist".

With funding from the Pfizer Foundation and the National Academy of Sciences, Virginia Commonwealth University has assembled more than 50 of the best videos from the public television series, Secrets of the Sequence to assist teachers in the application of genetic research across the biology curriculum. Each 8-10 minute video addresses a particular topic and is accompanied by classroom-tested lesson plans.

Stem Cell Resources contains information on tissue engineering, cloning, stem cell research and therapeutics, and ethical considerations.

Sumanas Animation contains animations showing the science behind stem cells, immunohistochemistry, DNA chip technology, in vitro fertilization, and pregnancy and paternity tests.

Teachers TryScience is a web site for teachers. This site provides free and engaging lessons, along with teaching strategies and resources, which are designed to spark students interest in science, technology, engineering and math (STEM). The site also provides collaboration tools for teachers to discuss and share effective instructional practices.

DNA Files contains high-quality recordings of radio programs on genetics, genetically-modified foods, gene therapy, biotechnology, transgenics, and more.

The Secret Life of Scientists and Engineers provides fun, interactive resources for kids of all ages, including videos that profile real scientists and their work. Students can send questions to the scientists featured. More than 30 profiles are available on the site.

The Strange New World of Nanoscience is an introductory video on Nanotechnology. The film was winner of the Best Short Film at the Scinema Science film festival 2010.

Understanding the Human Genome Project contains downloadable combinations of videos, discussion questions, and lesson plans on human genetics, genome sequencing, bioinformatics, variation and human history as well as ethical, legal, and social implications.

"What's for Lunch?" is a short, student-oriented video documentary on issues related to genetically-modified foods.

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Biotechnology Teacher Resources Online | North Carolina ...

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