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The stem cell hope : how stem cell medicine can change our lives
Name: The stem cell hope : how stem cell medicine can change our lives
Author: alice park
Pages: 122
Year: 2011
Language: English
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Cell Stem Cell Previews Oxidative Reductionist Approaches to Stem and Progenitor Cell Function Mark Noble, 1, *Chris Pro schel, 1 and Margot Mayer Pro schel 1 1 Department of Biomedical Genetics, University of Rochester Stem Cell and Regenerative Medicine Institute, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA *Correspondence:[email protected] DOI10.1016/j.stem.2010.12.005 Redox status is a critical modulator of stem and progenitor cell function. In this issue ofCell Stem Cell, Le Belle et al. (2011)demonstrate that oxidation promotes self renewal of neuroepithelial stem cells, revealing fascinating differences and surprising similarities with how redox pathways regulate glial progenitor cells.The status of being oxidized or reduced is one of the most fundamental regulators of cell function. It has become increasingly clear that small changes in redox status are critical in regulating the function of multiple signaling pathways and tran scription factors, that such regulation is central to normal cell function and not just in conditions of oxidative stress, and that both signaling molecules and tran scriptional regulators exert many of their effects through modulation of redox status. Thus, despite the existing focus on the regulation of stem/progenitor cell function by specific signaling and tran scriptional events, it could be argued that the regulation of these cells at the level of redox modulation may be of equal if not greater importance. A welcome new addition to the litera ture on redox regulation of precursor cell function is the current article by Kornblum and colleagues (Le Belle et al., 2011) that demonstrates the importance of reactive oxygen species (ROS) in regulating self renewal and neurogenesis in central nervous system (CNS) stem and progen itor cells. Their results provide highly convincing evidence that increases in oxidative status enhance neurosphere generation by neuroepithelial stem cells (NSCs) of the CNS. Specifically, exoge nous agents that elevate ROS levels increased production of neurospheres, one of the key in vitro assays for stem cell activity of NSCs. Freshly isolated cells from the subventricular zone (SVZ; the predominant location of stem cells in the CNS) that express stem cell antigens exhibit high levels of ROS, while stem cell antigen negative cells harbor less ROS. One key contributor to theseincreased ROS levels is NADPH oxidase(NOX), and pharmacological inhibition ofNOX inhibits neurosphere formation. Moreover, cells isolated from the SVZ of NOX2? /?mice showed lower ROS levels and diminished capacity for NSC self renewal and retention of multipotency during passaging in vitro. Brain derived neurotrophic factor (BDNF), which can further enhance neurosphere generation in cultures exposed to adequate levels of EGF and FGF, increased ROS levels inthesecells.Furthermore,NOXinhibition or treatment with the antioxidant and glutathione pro drug N acetyl L cysteine (NAC) inhibited the effects of BDNF on NSCs. BDNF was also not able to stimu late self renewal in cells isolated from NOX2 ?/? mice. One of the most striking aspects of the findingsofLeBelleetal.(2010)isthatthey represent, in many respects, a reverse image of previous studies that examined redox regulation of oligodendrocyte/ type 2 astrocyte progenitor cells (also known as oligodendrocyte precursor cells, and here abbreviated as O 2A/ OPCs). In O 2A/OPCs, it is the more reduced cells that exhibit enhanced self renewal properties, while cells that are relatively oxidized have a higher proba bility of differentiating into nondividing oligodendrocytes (Power et al., 2002; Smith et al., 2000). Moreover, increasing glutathione with NAC in O 2A/OPCs promotes self renewal, whereas expo sure to chemical pro oxidants inhibits cell division. Remarkably, despite the opposite effects of redox changes on NSC and O 2A/OPC proliferation and differentia tion, there are multiple similarities thatreveal certain common principles atwork. For example, in both cases, thecorrelation between redox status in vitro and in vivo is strongly conserved, such that NSCs freshly isolated from regions where they normally undergo more self renewal are more oxidized (Le Belle et al., 2011) and O 2A/OPCs isolated from developing regions of CNS in which self renewal occurs for extended periods are more reduced (Power et al., 2002; Smith et al., 2000). In addition, cells puri fied from the animal on the basis of their redox status exhibit the predicted differ ences in self renewal for both NSCs and O 2A/OPCs. Moreover, in both cases, cells more prone to self renewal exhibit some ability to maintain their redox set point when grown in conditions that would otherwise alter their redox state. In other words, NSCs remained relatively oxidized when grown in 4% (physio logical) O2 levels, and the more reduced O 2A/OPCs remained reduced when grown in 21% (atmospheric) O2. The presence of homeostatic regulation of redox set points suggests strongly that regulation of a particular redox balance is of critical importance in the function of stem/progenitor cells in the CNS. Common principles also are apparent when considering the essential nature of redox regulation as a mediator of the effects of signaling molecules relevant to NSC and O 2A/OPC function. In both cell types, cell signaling ligands that alter the balance between self renewal and differ entiation alter redox state in precisely the direction predicted by the effects on self renewal probability of chemical redox modulators. In NSCs, BDNF promotes self renewalandexposuretothiscytokineCell Stem Cell8, January 7, 2011 2011 Elsevier Inc.1


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makesthesecellsmoreoxidized.InO 2A/ OPCs,fibroblastgrowthfactor 2andneu rotrophin 3 enhance self renewal and make cells more reduced, while thyroid hormone and bone morphogenetic protein 4 promote differentiation and make cells more oxidized. Critically, in every case, inhibiting the redox changes caused by the signaling molecules abro gates their effects on self renewal and differentiation. Suchfindings make it clear that analysis of cell signaling function purely in terms of phosphorylation cascades, transcriptional regulation, etc., provides only a partial understanding of the means by which signaling regulates precursor cell function. In addition, it is clear for both O 2A/OPCs and NSCs the effects of redox modulation are quite specific (Li et al., 2007), lending support to the idea that rather than acting as a mere cofactor in general cell biological processes, redox state can act as a specific regulator of stem/progenitor cell function. The current findings on NSCs are not the only example in which being more oxidized enhances self renewal and/or division. In the CNS, hippocampal cells that give rise to neurons are stimulated to divide by oxidation (Limoli et al., 2004), as are a variety of other non CNS cells (Sauer et al., 2001). But when considering stem cells, it is important to consider the biological function of rapidly dividing cells. Outside of the earliest stages of development, stem cells are thought to exist mainly in a slowly dividing, ''quiescent'' state, and studies of hematopoietic stem cells (HSCs) suggest that oxidation is associated with the transition from quiescence to a rapidly dividing stage. This proliferative pool retains the capacity for multilineage reconstitution but loses the ability for long term, serial repopulation of the bone marrow (Kim et al., 1998), which is considered a gold standard functionalassay for self renewal. It is intriguing tospeculate whether the generation of rapidly dividing cells is a universal stem cell response to injury and whether the increased ROS production seen in most or all injuries might be a universal signal to stem cells to exit quiescence. But it is clear that even cells that find oxidation beneficial generate cells that have a re dox response more like O 2A/OPCS, as evidenced by the death of neurons in the same oxidative conditions that promoted their generation from NSCs (Le Belle et al., 2011). How are alterations in redox status translated into changes in self renewal and differentiation? In O 2A/OPCs, small increases in oxidative status cause acti vation of Fyn kinase, leading to activation of the ubiquitin ligase c Cbl and acceler ated degradation of its target proteins, including several critical receptor tyrosine kinases (RTKs) (Li et al., 2007). Loss of RTKs leads to suppression of down stream signaling through ERKs and Akt. In contrast, in NSCs, oxidative suppres sion of PTEN activity leads to elevated Akt activity, and the Akt pathway appears to be essential for NSC self renewal (Le Belle et al., 2011). But connections to other components of the cell cycle machinery still need to be made. It is also particularly intriguing that many of the signaling players identified thus far (e.g., PTEN, Fyn, c Cbl) are present in virtually all cell types, which raises the question of what regulatory network enables distinct outcomes in different cell types. Redox regulation of stem/progenitor cell function should also be considered carefully by the developing field of tissue repair by stem/progenitor cells. It is already clear that differences in redox status can be used to isolate cells of differing self renewal potential (Le Belle et al., 2011; Smith et al., 2000) and there are growing numbers of examples inwhich oxygen concentrations modulates stem/progenitor cell function (Mazumdar et al., 2009; Mohyedin et al., 2010). But will the redox status of the host also determine the ability of endogenous or transplanted stem/progenitor cells to carry out repair? Given that, in some populations, even a 15% increase in glutathione content causes a >1000% increase in cell survival (Mayer and Noble, 1994), relatively small metabolic fluctua tions may greatly change the outcome of experiments and clinical trials. Consid ering that the redox state is altered in almost every type of tissue injury, efforts to understand how the repair response of specific cell types may be altered by particular redox states may prove essen tialtoachievinganoptimalclinicalbenefit. REFERENCES Kim, M., Cooper, D., Hayes, S., and Spangrude, G. (1998). Blood91, 4106 4117. Le Belle, J.E., Orozco, N.M., Paucar, A.A., Saxe, J.P., Mottahedeh, J., Pyle, A.D., Wu, H., and Korn blum, H.I. (2011). Cell Stem Cell8, this issue, 59 71. Li, Z., Dong, T., Pro schel, C., and Noble, M. (2007). PLoS Biol.5, e35. 10.1371/journal.pbio.0050035. Limoli, C.L., Rola, R., Giedzinksi, E., Mantha, S., Huang, T. T., and Fike, J.R. (2004). Proc. Natl. Acad. Sci. USA101, 16052 16057. Mayer, M., and Noble, M. (1994). Proc. Natl. Acad. Sci. USA91, 7496 7500. Mazumdar, J., Dondeti, V., and Simon, M.C. (2009). J. Cell. Mol. Med.13, 4319 4328. Mohyedin, A., Garzo n Muvdi, T., and Quin ones Hinojosa, A. (2010). Cell Stem Cell6, 150 161. Power, J., Mayer Proschel, M., Smith, J., and Noble, M. (2002). Dev. Biol.245, 362 375. Sauer, H., Wartenberg, M., and Hescheler, J. (2001). Cell. Physiol. Biochem.11, 173 186. Smith, J., Ladi, E., Mayer Pro schel, M., and Noble, M. (2000). Proc. Natl. Acad. Sci. USA97, 10032 10037. Cell Stem Cell Previews 2Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc.


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Aging by Telomere Loss Can Be Reversed Bruno Bernardes de Jesus 1 and Maria A. Blasco 1, * 1 Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Melchor Ferna ndez Almagro 3, Madrid E 28029, Spain *Correspondence:[email protected] DOI10.1016/j.stem.2010.12.013 Recently inNature,Jaskelioff et al. (2010)demonstrated that multiple aging phenotypes in a mouse model of accelerated telomere loss can be reversed within 4 weeks of reactivating telomerase. This raises the major question of whether physiological aging, likely caused by a combination of molecular defects, may also be reversible. Accumulation of short/damaged telo meres with increasing age is considered one of the main sources of aging associ ated DNA damage responsible for the loss of regenerative potential in tissues and during systemic organismal aging (Harley et al., 1990; Flores et al., 2005). Mounting evidence suggests that telome rase is a longevity gene that functions by counteracting telomere attrition. Thus, telomerase deficient mice age prema turely, and telomerase overexpression results in extended longevity in mice (Tomas Loba et al., 2008). Moreover, human mutations in telomerase compo nents produce premature adult stem cell dysfunction and decreased longevity (Mitchell et al., 1999). Previous work had shown that restora tion of telomerase activity in mouse zygotes with critically short telomeres, owing to a deficiency in the telomerase RNA component (Terc),rescues critically short telomeres and chromosomal instability in the resulting mice (Samper et al., 2001). Restoration of telomerase activity in zygotes also pre vented the wide range of degenerative pathologies that would otherwise appear in telomerase deficient mice withcriticallyshorttelomeres, including bone marrow apla sia, intestinal atrophy, male germ line depletion, and adult stem cell dysfunction (Samper et al., 2001; Siegl Cachedenier et al., 2007), and resulted in a normal organismal life span (Siegl Cachedenier et al., 2007). Together, all the above find ings indicate that aging provoked by crit icaltelomereshorteningcanbeprevented or delayed by telomerase reactivation. From these grounds, reversion of aging caused by telomere loss was the next frontier. A recent study inNaturetakes an important step forward from these previous findings by using a new mouse model for telomerase deficiency, de signed to permit telomerase reactivation inadultmiceaftertelomere inducedaging phenotypes have been established (Jas kelioff et al., 2010). Specifically, DePinho and colleagues generated a knockin allele encoding a 4 OH tamoxifen (4 OHT) inducible mouse telomerase (TERT ER) underthecontroloftheTERTendogenous promoter. In the absence of tamoxifen, these mice exhibit premature appearance of aging pathologies and reduction in survival(Figure1).Thesemicephenocopypreviously describedTerc deficient mice, which highlights that elongation of short telomeres by telomerase is the main mechanism bywhich telomerase protects from aging pathologies. Importantly, 4 weeks of tamoxifen treatment to induce TERT re expression in adult TERT ER mice with clear signs of premature aging was sufficient to extend their telomeres and rescue telomeric DNA damage signaling and associated check point responses. Dramatically, tamox ifen induced TERT re expression also led to resumption of proliferation in quies cent cultured cells and eliminated the degenerative phenotypes across multiple organs,includingtestis,spleen,andintes tines (Figure 1). Reactivation of telome rase also ameliorated the decreased survival of TERT ER mice. These findings represent an important advance in the aging field, as they show that aging induced by telomere loss can be reversed in a broad range of tissues and cell types, including neuronal function. Looking to the future, the next key question is to what extent natural, physiological aging is caused by the pres ence of critically short telo meres and, consequently, to what extent telomere restora tion will be able to reverse physiological aging. In this re gard, other recent findings support the ideathattelomere shortening does impact natural mouse aging. On one hand, despite the long standing belief that mouse aging was not linked to telo me re shortening given thatFigure 1. Antiaging Effects of Telomerase Schematic showing the major findings ofJaskelioff et al. (2010). Telomerase reactivation in late generation telomerase deficient mice (G4 TERT ER ) could revert some of the aging phenotypes observed, demonstrating the regenera tive potential capacity of different tissues. Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc.3 Cell Stem Cell Previews


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mice are born with very long telomeres much longer than human telomeres mouse telomeres do suffer extensive shortening associated with aging (Flores etal.,2008).Inparticular,whilemousecells maintain relatively long telomeres during their first year of life, there is a dramatic loss of telomeric sequences at 2 years of age, even in various stem cell populations, and this change is concomitant with the loss of regenerative capacity associated with mouse aging. In addition, telome rase deficient mice from the first genera tion (G1Terc ?/? )exhibitasignificant decrease in median and maximum longevity and a higher incidence of age related pathologies and stemcell dysfunc tion compared with wild type mice (Flores et al., 2005; Garcia Cao et al., 2006), indi cating that, as in humans, telomerase activity is rate limiting for natural mouse longevity and aging. These results suggest that strategies aimed to increase telome rase activity may delay natural mouse aging. Further supporting this notion, it was recently shown that overexpression of TERT in the context of mice engineered to be cancer resistant owe to increaseexpression of tumor suppressor genes(Sp53/Sp16/SARF/TgTERT mice) was sufficient to decrease telomere damage withage,delayaging,andincreasemedian longevity by 40% (Tomas Loba et al., 2008). However, it remains to be seen whether telomerase reactivation late in life would be sufficient to delay natural mouse aging and extend mouse longevity without increasing cancer incidence. In summary, these proof of principle studies using genetically modified mice are likely to encourage the development of targeted therapeutic strategies based on reactivation of telomerase function. Indeed, small molecule telomerase acti vators have been reported recently and have demonstrated some preliminary health span beneficial effects in humans (Harley et al., 2010). Identifying drugable targets and candidate activators clearly opens a new window for the treatment of age associated degenerative diseases. REFERENCES Flores, I., Cayuela, M.L., and Blasco, M.A. (2005). Science309, 1253 1256.Flores, I., Canela, A., Vera, E., Tejera, A., Cotsare lis, G., and Blasco, M.A. (2008). Genes Dev.22, 654 667. Garcia Cao, I., Garcia Cao, M., Tomas Loba, A., Martin Caballero, J., Flores, J.M., Klatt, P., Blasco, M.A., and Serrano, M. (2006). EMBO Rep.7, 546 552. Harley, C.B., Futcher, A.B., and Greider, C.W. (1990). Nature345, 458 460. Harley, C.B., Liu, W., Blasco, M., Vera, E., Andrews, W.H., Briggs, L.A., and Raffaele, J.M. (2010). Rejuvenation Res.14, in press. Published online September 7, 2010. 10.1089/rej.2010.1085. Jaskelioff, M., Muller, F.L., Paik, J.H., Thomas, E., Jiang, S., Adams, A.C., Sahin, E., Kost Alimova, M., Protopopov, A., Cadinanos, J., et al. (2010). Nature. 10.1038/nature09603. Mitchell, J.R., Wood, E., and Collins, K. (1999). Nature402, 551 555. Samper, E., Flores, J.M., and Blasco, M.A. (2001). EMBO Rep.2, 800 807. Siegl Cachedenier, I., Flores, I., Klatt, P., and Blasco, M.A. (2007). J. Cell Biol.179, 277 290. Tomas Loba, A., Flores, I., Fernandez Marcos, P.J., Cayuela, M.L., Maraver, A., Tejera, A., Borras, C., Matheu, A., Klatt, P., Flores, J.M., et al. (2008). Cell135, 609 622. HGPS Derived iPSCs For The Ages Tom Misteli 1, * 1 National Cancer Institute, NIH, Bethesda, MD 20892, USA *Correspondence:[email protected] DOI10.1016/j.stem.2010.12.014 In this issue ofCell Stem Cell,Zhang et al. (2011)generate patient derived iPSCs for one of the major prema tureagingdiseases,Hutchinson GilfordProgeriaSyndrome(HGPS).Thesecellsareamuch needednewtool to study HGPS, and their use may lead to novel insights into mechanisms of aging. Some problems in biology are more difficult to study than others. Human aging is certainly one of them. Most conclusions regarding molecular mecha nism ofhuman aging rely on mere correla tion, and direct experimental testing is generally not feasible. One approach to dissect the molecular basis of human aging is to study naturally occurring premature aging disorders. One of the most dramatic and prominent of suchdiseases is Hutchinson Gilford Progeria Syndrome (HGPS).Zhang et al. (2011) now report the generation of induced pluripotent stem cells (iPSCs) from HGPS cells, providing a powerful new tool to unravel the molecular and physio logical mechanisms of premature and normal aging. HGPS is a truly remarkable disease in many ways. To start with, it affects an unusually wide spectrum of tissues andleadstothedevelopmentofhighlydiverse symptoms ranging from depletion of subcutaneous fat to loss of hair and tendon contractures. The diversity of affected tissues pointed early on to stem cell defects as a likely disease mecha nism. Most relevant in patients are vascular defects and recurring strokes, which invariably are fatal in patients in their mid to late teens (Hennekam, 2006). The disease is exceedingly rare 4Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc. Cell Stem Cell Previews


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mice are born with very long telomeres much longer than human telomeres mouse telomeres do suffer extensive shortening associated with aging (Flores etal.,2008).Inparticular,whilemousecells maintain relatively long telomeres during their first year of life, there is a dramatic loss of telomeric sequences at 2 years of age, even in various stem cell populations, and this change is concomitant with the loss of regenerative capacity associated with mouse aging. In addition, telome rase deficient mice from the first genera tion (G1Terc ?/? )exhibitasignificant decrease in median and maximum longevity and a higher incidence of age related pathologies and stemcell dysfunc tion compared with wild type mice (Flores et al., 2005; Garcia Cao et al., 2006), indi cating that, as in humans, telomerase activity is rate limiting for natural mouse longevity and aging. These results suggest that strategies aimed to increase telome rase activity may delay natural mouse aging. Further supporting this notion, it was recently shown that overexpression of TERT in the context of mice engineered to be cancer resistant owe to increaseexpression of tumor suppressor genes(Sp53/Sp16/SARF/TgTERT mice) was sufficient to decrease telomere damage withage,delayaging,andincreasemedian longevity by 40% (Tomas Loba et al., 2008). However, it remains to be seen whether telomerase reactivation late in life would be sufficient to delay natural mouse aging and extend mouse longevity without increasing cancer incidence. In summary, these proof of principle studies using genetically modified mice are likely to encourage the development of targeted therapeutic strategies based on reactivation of telomerase function. Indeed, small molecule telomerase acti vators have been reported recently and have demonstrated some preliminary health span beneficial effects in humans (Harley et al., 2010). Identifying drugable targets and candidate activators clearly opens a new window for the treatment of age associated degenerative diseases. REFERENCES Flores, I., Cayuela, M.L., and Blasco, M.A. (2005). Science309, 1253 1256.Flores, I., Canela, A., Vera, E., Tejera, A., Cotsare lis, G., and Blasco, M.A. (2008). Genes Dev.22, 654 667. Garcia Cao, I., Garcia Cao, M., Tomas Loba, A., Martin Caballero, J., Flores, J.M., Klatt, P., Blasco, M.A., and Serrano, M. (2006). EMBO Rep.7, 546 552. Harley, C.B., Futcher, A.B., and Greider, C.W. (1990). Nature345, 458 460. Harley, C.B., Liu, W., Blasco, M., Vera, E., Andrews, W.H., Briggs, L.A., and Raffaele, J.M. (2010). Rejuvenation Res.14, in press. Published online September 7, 2010. 10.1089/rej.2010.1085. Jaskelioff, M., Muller, F.L., Paik, J.H., Thomas, E., Jiang, S., Adams, A.C., Sahin, E., Kost Alimova, M., Protopopov, A., Cadinanos, J., et al. (2010). Nature. 10.1038/nature09603. Mitchell, J.R., Wood, E., and Collins, K. (1999). Nature402, 551 555. Samper, E., Flores, J.M., and Blasco, M.A. (2001). EMBO Rep.2, 800 807. Siegl Cachedenier, I., Flores, I., Klatt, P., and Blasco, M.A. (2007). J. Cell Biol.179, 277 290. Tomas Loba, A., Flores, I., Fernandez Marcos, P.J., Cayuela, M.L., Maraver, A., Tejera, A., Borras, C., Matheu, A., Klatt, P., Flores, J.M., et al. (2008). Cell135, 609 622. HGPS Derived iPSCs For The Ages Tom Misteli 1, * 1 National Cancer Institute, NIH, Bethesda, MD 20892, USA *Correspondence:[email protected] DOI10.1016/j.stem.2010.12.014 In this issue ofCell Stem Cell,Zhang et al. (2011)generate patient derived iPSCs for one of the major prema tureagingdiseases,Hutchinson GilfordProgeriaSyndrome(HGPS).Thesecellsareamuch needednewtool to study HGPS, and their use may lead to novel insights into mechanisms of aging. Some problems in biology are more difficult to study than others. Human aging is certainly one of them. Most conclusions regarding molecular mecha nism ofhuman aging rely on mere correla tion, and direct experimental testing is generally not feasible. One approach to dissect the molecular basis of human aging is to study naturally occurring premature aging disorders. One of the most dramatic and prominent of suchdiseases is Hutchinson Gilford Progeria Syndrome (HGPS).Zhang et al. (2011) now report the generation of induced pluripotent stem cells (iPSCs) from HGPS cells, providing a powerful new tool to unravel the molecular and physio logical mechanisms of premature and normal aging. HGPS is a truly remarkable disease in many ways. To start with, it affects an unusually wide spectrum of tissues andleadstothedevelopmentofhighlydiverse symptoms ranging from depletion of subcutaneous fat to loss of hair and tendon contractures. The diversity of affected tissues pointed early on to stem cell defects as a likely disease mecha nism. Most relevant in patients are vascular defects and recurring strokes, which invariably are fatal in patients in their mid to late teens (Hennekam, 2006). The disease is exceedingly rare 4Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc. Cell Stem Cell Previews


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with only about 200 patients in the world at any time, making access to relevant tissues very difficult. HGPS is also re markable in how much we know about its molecular and cellular basis. HGPS is caused by a mutation in theLMNAgene encoding the intermediate filament proteins lamin A and C, key architectural components of the cell nucleus and both involved in higher order genome organi zation (Worman et al., 2010). The disease mutation leads to activation of a cryptic splice site inLMNAand the production of a dominant gain of function isoform of lamin A, referred to as progerin. This protein is permanently farnesylated at its C terminus and accumulates in the nuclear lamina, where it disrupts normal lamina function. Progerin is not only relevant to HGPS, but also to normal aging, because the cryptic splice site which creates progerin is also used at low frequency in healthy individuals and progerin can be found in normal tissues (Scaffidi and Misteli, 2006). Further parallels between HGPS and normal aging are suggested, given that several cellular defects such as loss of epigenetic marks and increased DNA damage are observed in both settings. In addition, HGPS patients and normally aged individuals exhibit similar vascular defects. Due to the rarity of the disease and the fragility of the patients it is diffi cult, however, to obtain relevant biolog ical materials for molecular analysis, and much of what we know about the disease"s mechanisms comes from cul tured skin cells and animal models. The generation of HGPS derived iPSCs now reported byZhang et al. (2011)now provides a much needed source for tissue specific cell lines with which to probe the effect of progerin on tissue function and differentiation. The HGPS derived iPSCs were gener ated from patient skin fibroblasts using the standard Yamanaka method (Zhang et al., 2011). The derived cells appeared pluripotent since they form teratomas and exhibit gene expression profiles akin to established human embryonic stem cell (hESC) lines. Interestingly, though, the efficiency of iPSC generation from HGPS patient cells was lower than from wild type control cells. This might be due, as the authors suggest, to early onset of senescence in HGPS cells, but it might also have something to do withan inhibitory role of progerin on the large scale chromatin reorganization required during reprogramming. We know that lamins tether chromatin to the periphery and clamp it down into hetero chromatin and that progerin solidifies the normally dynamic nuclear lamina (Dahl et al., 2006). ESCs are one of few human cell types that do not express lamins A and C, and at the same time, they lack heterochromatin, possibly as a means to maintain broad genome plasticity. It is conceivable that the presence of progerin in HGPS cells prevents the dynamic reorganization of chromatin required for efficient reprogramming. The derivation of HGPS iPSCs is of significant practical importance. The described cells are able to differentiate into five lineages, including vascular smooth muscle cells (VSMCs) and mesenchymal stem cells (MSCs) (Zhang et al., 2011), confirming their multipo tency. These cells now offer a useful experimental system to probe the effect of progerin on the differentiation of various cell lineages, something that could not be done before because of the inability to obtain tissue samples from patients. These cells also open the door to performing critical experiments, such as transplantation of HGPS derived MSCs into the vasculature of animal models to probe the physiological mech anisms that participate in the vascular defects experienced by HGPS patients. The HGPS iPSCs,and theirderivatives, will also be useful for drug discovery. At present, the only clinical strategy for HGPS is farnesyltransferase inhibitors (FTIs), which prevent the addition of the C terminal farnesyl group on progerin (Capell and Collins, 2006). While FTIs have been shown to reverse cellular phenotypes and have a positive effect on vasculature and on extension of life span in animal models, the nonspecific nature of the drug might become limiting in clinical applications. Lineage differenti ated cell lines derived from HGPS iPSCs will provide ample and well controlled biological materialsforthesearchofnovel drugs in high throughput screens. Although the HGPS derived iPSCs appear to differentiate normally in vitro, they are functionally compromised, pro viding some insights into disease mecha nism (Zhang et al., 2011). HGPS iPSC derived cells are hypersensitive to variousformsof stress. Survival of HGPS iPSC derived VSMCs was significantly reduced under hypoxic conditions or when sub jected to extended electrical stimulation. The latter is potentially relevant to their pathological function because VSMCs undergo extensive mechanical stress in vivo due to the pulsing of the vascula ture, and the reduced survival and prolif eration observed in vitro may suggest increased cell death in the vasculature of HGPS patients. HGPS iPSC derived MSCs were also functionally compro mised in vivo. When transplanted into an ischemic hind limb muscle, they were unable to prevent necrosis, whereas MSCs derived in parallel from control iPSCs did. This failure may be due to the inability of HGPS derived MSCs to replace vascular cells that are removed due to their normal turnover and/or the poor survival of these cells in the hypoxic environment of the muscle. Although it remains unclear why exactly the HGPS iPSC derived MSCs failed to rescue these defects, it is tempting to consider that MSC transplantation may offer a novel therapeutic option for HGPS. An intriguing, albeit distant, goal may be the generation of patient derived MSCs in which theLMNAmutation has been corrected using recombination based approaches. Theseobservationsonmuscleregener ation are also directly relevant to our thinking about normal aging. Loss of regeneration capacity has become a pre vailing, albeit quite obvious, model for aging (Sharpless and DePinho, 2007). If tissue cells, and particularly stem cells, which are lost from a tissue due to normal turnover, are not replaced efficiently, tissues will, of course, deteriorate. It ap pears that in the case of HGPS, and likely in normal aging, tissue stem cells become increasinglyunabletokeepupwithregen eration of lost tissue cells. This pattern may arise for several reasons. Tissue stem cell numbers may be reduced due to increased apoptosis, in the case of HGPS possibly due to their inability to cope with stress, for example, under hypoxic conditions in tissues. In addition, tissue stem cells might fail to self renew, or they may produce fewer and function allyimpairedoffspring.TheHGPS derived iPSCsshouldbeusefulinfurtherresolving therelevanceofthesevariouspathwaysto organismal aging. Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc.5 Cell Stem Cell Previews


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HGPS is an extraordinary disease, and the generation of patient derived iPSCs is a significant milestone. This step continues the remarkable progress madeinthelastfewyears.Afterdiscovery of the disease causing gene in 2003, it only took four years to initiate several clinical trials. Much has been learnt along the wayaboutthe biologyofHGPS andits relevancetonormalaging.Thegeneration of iPSCs fromHGPS patientsnow heralds another wave of rapid progress withimplications for HGPS disease mecha nisms,foragingingeneral,andpotentially as a tool to develop novel strategies to combat vascular disease. REFERENCES Capell, B.C., and Collins, F.S. (2006). Nat. Rev. Genet.7, 940 952. Dahl, K.N., Scaffidi, P., Islam, M.F., Yodh, A.G., Wilson, K.L., and Misteli, T. (2006). Proc. Natl. Acad. Sci. USA103, 10271 10276.Hennekam,R.C.(2006).Am.J.Med.Genet.A.140, 2603 2624. Scaffidi, P., and Misteli, T. (2006). Science312, 1059 1063. Sharpless, N.E., and DePinho, R.A. (2007). Nat. Rev. Mol. Cell Biol.8, 703 713. Worman, H.J., Ostlund, C., and Wang, Y. (2010). Cold Spring Harb. Perspect. Biol.2, a000760. Zhang, J.L., Zhu, Q., Zhou, G., Sui, F., Tan, L., Mutalif, A., Navasankari, R., Zhang, Y., Tse, H. F., Stewart, C., et al. (2011). Cell Stem Cell8, this issue, 31 45. A Roundabout Way to the Niche Kateri Moore 1,2, * 1 Departments of Gene and Cell Medicine 2 Department of Developmental and Regenerative Biology Mount Sinai School of Medicine, New York, NY 10029, USA *Correspondence:[email protected] DOI10.1016/j.stem.2010.12.011 A new player in hematopoietic stem cell (HSC) niche interactions is introduced in this issue ofCell Stem Cell. Smith Berdanetal.(2010)demonstratethatRobo4isinvolvedinHSCengraftmentandmobilizationanddoes so in cooperation with Cxcr4 to guide stem cells to and secure them in the niche. Bone marrow (BM) transplantation has been used for treatment of hematopoietic disorders for some fifty years and repre sents a paradigm for all future stem cell therapies. A number of cytokines, espe ciallygranulocytecolony stimulatingfactor (G CSF), are known to mobilize hemato poietic stem and progenitor cells (HSPCs) from their BM niches into the peripheral blood (PB) (Papayannopoulou and Scad den, 2008). Indeed, mobilization is the preferred method for obtaining transplant able HSC. Despite the number of currently available HSPC mobilizing agents, a significant number of donors mobilize poorly. Therefore, identifying novel and more efficient mobilization approaches is of paramount clinical importance. Understanding the molecular frame work of how the niche regulates retention and release of stem cells provides the groundonwhichtobasealternativemobi lization strategies. The basic processes of transplantation are homing to, engraft ment in, and retention of HSCs in the niche. Mobilization may thus be under stood as the process of breaking the bonds of stem cell retention in the BM niche or enhancement of the existing means that allow HSCs to enter the PB. The cellular milieu and molecular mecha nisms that mediate these processes are startingtoberevealedbut,atbest,remain poorly understood (Garrett and Emerson, 2009). The Cxcr4/Cxcl12 axis has been identifiedascriticallyimportantinhoming, engraftment,andretentionintheBM(Lap idotetal.,2005).Previousworkhasshown that the Cxcr4 antagonist AMD3100 can mobilize both mouse and human HSPCs and has found use clinically as an adjunct therapy for poor G CSF mobilizers (Brox meyer et al., 2005). In this issue ofCell Stem Cell, Smith Berdan et al. show that Roundabout 4 (Robo4), a neuronal guid ance molecule, regulates engraftment and mobilization and, in cooperation with Cxcr4, localizes HSCs to the niche. Previous profiling studies by the senior author had revealed that Robo4 was ex pressed at high levels in long term HSCs (Forsberg et al., 2005). In the presentwork, the authors show that Robo4 becomes downregulated upon differenti ation, consistent with the observations of Shibata et al., who also demonstrated that repopulating cells segregated to the Robo4 + fraction of HSPCs (Shibata et al., 2009). Notably, Smith Berdan et al. also foundthatRobo4expressionwasdramat ically downregulated in mobilized HSCs. To determine a functional role for Robo4 in HSCs, the authors investigated Robo4 knockout mice.Robo4 ?/? mice appear normal but have defects in vascular integ rity and angiogenesis (Jones et al., 2008). Ananalysisofthestemcellcompartments revealed thatRobo4 ?/? mice had a spe cific decrease of HSCs in the BM with a reciprocal increase in PB, suggesting poor BM retention. Upon transplantation, Robo4 ?/? HSCs engrafted poorly, but those that did engraft contributed to a normal spectrum of blood cell lineages. In addition, the ability ofRobo4 ?/? HSC tomake spleencolonies was normal,sug gesting that the engraftment defect was likely because of a specific impairment of 6Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc. Cell Stem Cell Previews


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HGPS is an extraordinary disease, and the generation of patient derived iPSCs is a significant milestone. This step continues the remarkable progress madeinthelastfewyears.Afterdiscovery of the disease causing gene in 2003, it only took four years to initiate several clinical trials. Much has been learnt along the wayaboutthe biologyofHGPS andits relevancetonormalaging.Thegeneration of iPSCs fromHGPS patientsnow heralds another wave of rapid progress withimplications for HGPS disease mecha nisms,foragingingeneral,andpotentially as a tool to develop novel strategies to combat vascular disease. REFERENCES Capell, B.C., and Collins, F.S. (2006). Nat. Rev. Genet.7, 940 952. Dahl, K.N., Scaffidi, P., Islam, M.F., Yodh, A.G., Wilson, K.L., and Misteli, T. (2006). Proc. Natl. Acad. Sci. USA103, 10271 10276.Hennekam,R.C.(2006).Am.J.Med.Genet.A.140, 2603 2624. Scaffidi, P., and Misteli, T. (2006). Science312, 1059 1063. Sharpless, N.E., and DePinho, R.A. (2007). Nat. Rev. Mol. Cell Biol.8, 703 713. Worman, H.J., Ostlund, C., and Wang, Y. (2010). Cold Spring Harb. Perspect. Biol.2, a000760. Zhang, J.L., Zhu, Q., Zhou, G., Sui, F., Tan, L., Mutalif, A., Navasankari, R., Zhang, Y., Tse, H. F., Stewart, C., et al. (2011). Cell Stem Cell8, this issue, 31 45. A Roundabout Way to the Niche Kateri Moore 1,2, * 1 Departments of Gene and Cell Medicine 2 Department of Developmental and Regenerative Biology Mount Sinai School of Medicine, New York, NY 10029, USA *Correspondence:[email protected] DOI10.1016/j.stem.2010.12.011 A new player in hematopoietic stem cell (HSC) niche interactions is introduced in this issue ofCell Stem Cell. Smith Berdanetal.(2010)demonstratethatRobo4isinvolvedinHSCengraftmentandmobilizationanddoes so in cooperation with Cxcr4 to guide stem cells to and secure them in the niche. Bone marrow (BM) transplantation has been used for treatment of hematopoietic disorders for some fifty years and repre sents a paradigm for all future stem cell therapies. A number of cytokines, espe ciallygranulocytecolony stimulatingfactor (G CSF), are known to mobilize hemato poietic stem and progenitor cells (HSPCs) from their BM niches into the peripheral blood (PB) (Papayannopoulou and Scad den, 2008). Indeed, mobilization is the preferred method for obtaining transplant able HSC. Despite the number of currently available HSPC mobilizing agents, a significant number of donors mobilize poorly. Therefore, identifying novel and more efficient mobilization approaches is of paramount clinical importance. Understanding the molecular frame work of how the niche regulates retention and release of stem cells provides the groundonwhichtobasealternativemobi lization strategies. The basic processes of transplantation are homing to, engraft ment in, and retention of HSCs in the niche. Mobilization may thus be under stood as the process of breaking the bonds of stem cell retention in the BM niche or enhancement of the existing means that allow HSCs to enter the PB. The cellular milieu and molecular mecha nisms that mediate these processes are startingtoberevealedbut,atbest,remain poorly understood (Garrett and Emerson, 2009). The Cxcr4/Cxcl12 axis has been identifiedascriticallyimportantinhoming, engraftment,andretentionintheBM(Lap idotetal.,2005).Previousworkhasshown that the Cxcr4 antagonist AMD3100 can mobilize both mouse and human HSPCs and has found use clinically as an adjunct therapy for poor G CSF mobilizers (Brox meyer et al., 2005). In this issue ofCell Stem Cell, Smith Berdan et al. show that Roundabout 4 (Robo4), a neuronal guid ance molecule, regulates engraftment and mobilization and, in cooperation with Cxcr4, localizes HSCs to the niche. Previous profiling studies by the senior author had revealed that Robo4 was ex pressed at high levels in long term HSCs (Forsberg et al., 2005). In the presentwork, the authors show that Robo4 becomes downregulated upon differenti ation, consistent with the observations of Shibata et al., who also demonstrated that repopulating cells segregated to the Robo4 + fraction of HSPCs (Shibata et al., 2009). Notably, Smith Berdan et al. also foundthatRobo4expressionwasdramat ically downregulated in mobilized HSCs. To determine a functional role for Robo4 in HSCs, the authors investigated Robo4 knockout mice.Robo4 ?/? mice appear normal but have defects in vascular integ rity and angiogenesis (Jones et al., 2008). Ananalysisofthestemcellcompartments revealed thatRobo4 ?/? mice had a spe cific decrease of HSCs in the BM with a reciprocal increase in PB, suggesting poor BM retention. Upon transplantation, Robo4 ?/? HSCs engrafted poorly, but those that did engraft contributed to a normal spectrum of blood cell lineages. In addition, the ability ofRobo4 ?/? HSC tomake spleencolonies was normal,sug gesting that the engraftment defect was likely because of a specific impairment of 6Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc. Cell Stem Cell Previews


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Robo4 ?/? HSCs to home, engraft, and remain in the BM. On the basis of these results, the Fors berg group hypothesized that Robo4 mediates HSC adhesion to the niche and that downregulation of Robo4 was a crit ical step enabling exit from the niche to the bloodstream. Consistent with this idea, the authors predicted that mobiliza tion induced by G CSF treatment would be elevated in Robo4 null mice. Instead, they found thatRobo4 ?/? HSCs were delayed in their ability to mobilize in response to G CSF. Smith Berdan et al. next examined the well known Cxcr4/ Cxcl12 axis and found that Cxcr4 expres sion in HSCs and Cxcl12/Sdf1 expression in stromal cells was elevated inRobo4 ?/? mice. Thus, a compensatory upregulation of the Cxcr4/Cxcl12 axis likely explains whyRobo4 ?/? HSCs were slower to mobilize. Mobilization experiments using AMD3100, a Cxcr4 antagonist, in con junction with G CSF or as the sole mobili zation agent, revealed that HSCs were specifically mobilized at higher levels in Robo4 ?/? mice. In order to test whether inhibition of the Cxcr4/Cxcl12 axis specif ically affects stem cell homing, HSCs were pretreated with AMD3100 before transplantation. HSCs from both strains homed less efficiently to BM after AMD3100 pretreatment but even less so when lacking Robo4, suggesting that Robo4 cooperates with Cxcr4 in stem cell homing. Taken together, these results suggest that a Robo4 antagonist would aid in specific mobilization of HSCs into thebloodstream andmayhavea potential clinical use in combination with otheragents. As such, these experiments pro vide enticing evidence for a novel path way in stem cell homing, engraftment, and mobilization from the niche. The findings of Smith Brennan et al. point to an exciting new line of investiga tion in stem/niche cell interactions with many questions to be probed in future work. At the forefront of these questions is whether the pattern of Robo4 expres sionin human HSCs mimics that in mouse and whether nongenetic approaches targeting Robo4 would be useful for mobilization and purification of HSCs. Mechanistically, the reciprocal loss of Robo4 and the upregulation of the Cxcr4/Cxcl12 axis remain to be defined. Is there a point where the two pathways intersect in their downstream signaling? Of interest, Robo4 is expressed in endo thelium and functions in vascular sprout ing upon activation by its ligand Slit2. It will be interesting to determine if Robo4 in this context acts via Slit2 and if there is an additional coreceptor. Activated Robo4 also stabilizes the vascular network through inhibition of endothelial permeability (Jones et al., 2008). Thus, how loss of Robo4 affects the endothelial function will be an important topic to address in future studies. Finally, where are the Robo4 + HSC in the BM normally localized and to where do they home? Osteoblasts upregulate the expression of Slit2 after 5 FU treatment (Shibata et al., 2009), and Slit2 expression has very recently been found in the extramural cells surrounding endothelium in devel oping mammary tissue (Marlow et al., 2010). It would be very interesting if Slit2expression were found in the Cxcl12 abundant reticular (CAR) cells that sur round endothelium, localize near the endosteum, and are thought to play a role in the stem cell niche (Sugiyama et al., 2006). Indeed, it should be very revealing to pursue this roundabout way into and out of the niche. REFERENCES Broxmeyer, H.E., Orschell, C.M., Clapp, D.W., Hangoc, G., Cooper, S., Plett, P.A., Liles, W.C., Li, X., Graham Evans, B., Campbell, T.B., et al. (2005). J. Exp. Med.201, 1307 1318. Forsberg, E.C., Prohaska, S.S., Katzman, S., Heffner, G.C., Stuart, J.M., and Weissman, I.L. (2005). PLoS Genet.1, e28. Garrett, R.W., andEmerson, S.G. (2009). Cell Stem Cell4, 503 506. Jones, C.A., London, N.R., Chen, H., Park, K.W., Sauvaget, D., Stockton, R.A., Wythe, J.D., Suh, W., Larrieu Lahargue, F., Mukouyama, Y.S., et al. (2008). Nat. Med.14, 448 453. Lapidot, T., Dar, A., and Kollet, O. (2005). Blood 106, 1901 1910. Marlow, R., Binnewies, M., Sorensen, L.K., Mon ica, S.D., Strickland, P., Forsberg, E.C., Li, D.Y., and Hinck, L. (2010). Proc. Natl. Acad. Sci. USA 107, 10520 10525. Papayannopoulou, T., and Scadden, D.T. (2008). Blood111, 3923 3930. Shibata, F., Goto Koshino, Y., Morikawa, Y., Ko mori,T.,Ito,M.,Fukuchi,Y.,Houchins,J.P.,Tsang, M., Li, D.Y., Kitamura, T., et al. (2009). Stem Cells 27, 183 190. Smith Berdan, S., Nguyen, A., Hassanein, D., Zim mer, M., Ugarte, F., Ciriza, J., Li, D., Garc a Ojeda, M., Hinck, L., and Forsberg, C. (2010). Cell Stem Cell8, this issue, 72 83. Sugiyama, T., Kohara, H., Noda, M., and Naga sawa, T. (2006). Immunity25, 977 988. Cell Stem Cell8, January 7, 2011 2011 Elsevier Inc.7 Cell Stem Cell Previews


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