| 1. |
- Mononen, Mimmi, et al.
(författare)
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Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage
- 2020
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Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
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Tidskriftsartikel (refereegranskat)abstract
- A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.
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| 2. |
- Xu, Jiejia, et al.
(författare)
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Genome‐wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors
- 2020
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Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
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Tidskriftsartikel (refereegranskat)abstract
- Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.
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| 3. |
- Adler, Andrew, et al.
(författare)
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Transsynaptic tracing and its emerging use to assess graftreconstructed neural circuits
- 2020
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 38:6, s. 716-726
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Tidskriftsartikel (refereegranskat)abstract
- Fetal neural progenitor grafts have been evaluated in preclinical animal models of spinal cord injury and Parkinson’s disease for decades, but the initial reliance on primary tissue as a cell source limited the scale of their clinical translatability. With the development of robust methods to differentiate human pluripotent stem cells to specific neural subtypes, cell replacement therapy holds renewed promise to treat a variety of neurodegenerative diseases and injuries at scale. As these cell sources are evaluated in preclinical models, new transsynaptic tracing methods are making it possible to study the connectivity between host and graft neurons with greater speed and detail than was previously possible. To date, these studies have revealed that widespread, long-lasting, and anatomically-appropriate synaptic contacts are established between host and graft neurons, as well as new aspects of host-graft connectivity which may be relevant to clinical cell replacement therapy. It is not yet clear, however, whether the synaptic connectivity between graft and host neurons is as celltype specific as it is in the endogenous nervous system, or whether that connectivity is responsible for the functional efficacy of cell replacement therapy. Here, we review evidence suggesting that the new contacts established between host and graft neuronsmay indeed be cell-type specific, and how transsynaptic tracing can be used inthe future to further elucidate the mechanisms of graft-mediated functional recovery in spinal cord injury and Parkinson’s disease.
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| 4. |
- Aldskogius, Håkan, 1943-, et al.
(författare)
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Regulation of boundary cap neural crest stem cell differentiation after transplantation
- 2009
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 27:7, s. 1592-1603
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Tidskriftsartikel (refereegranskat)abstract
- Success of cell replacement therapies for neurological disorders will dependlargely on the optimization of strategies to enhance viability and control thedevelopmental fate of stem cells after transplantation. Once transplanted,stem/progenitor cells display a tendency to maintain an undifferentiatedphenotype or differentiate into inappropriate cell types. Gain and loss offunction experiments have revealed key transcription factors which drivedifferentiation of immature stem/progenitor cells toward more mature stages andeventually to full differentiation. An attractive course of action to promotesurvival and direct the differentiation of transplanted stem cells to a specific cell type would therefore be to force expression of regulatory differentiationmolecules in already transplanted stem cells, using inducible gene expressionsystems which can be controlled from the outside. Here, we explore thishypothesis by employing a tetracycline gene regulating system (Tet-On) to drivethe differentiation of boundary cap neural crest stem cells (bNCSCs) toward asensory neuron fate after transplantation. We induced the expression of the keytranscription factor Runx1 in Sox10-expressing bNCSCs. Forced expression of Runx1strongly increased transplant survival in the enriched neurotrophic environmentof the dorsal root ganglion cavity, and was sufficient to guide differentiationof bNCSCs toward a nonpeptidergic nociceptive sensory neuron phenotype both invitro and in vivo after transplantation. These findings suggest that exogenousactivation of transcription factors expression after transplantation instem/progenitor cell grafts can be a constructive approach to control theirsurvival as well as their differentiation to the desired type of cell and thatthe Tet-system is a useful tool to achieve this.
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| 5. |
- Ameri, Jacqueline, et al.
(författare)
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FGF2 Specifies hESC-Derived Definitive Endoderm into Foregut/Midgut Cell Lineages in a Concentration-Dependent Manner.
- 2010
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28, s. 45-56
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Tidskriftsartikel (refereegranskat)abstract
- Fibroblast growth factor (FGF) signaling controls axis formation during endoderm development. Studies in lower vertebrates have demonstrated that FGF2 primarily patterns the ventral foregut endoderm into liver and lung, whereas FGF4 exhibits broad anterior-posterior and left-right patterning activities. Furthermore, an inductive role of FGF2 during dorsal pancreas formation has been shown. However, whether FGF2 plays a similar role during human endoderm development remains unknown. Here, we show that FGF2 specifies hESC-derived definitive endoderm (DE) into different foregut lineages in a dosage-dependent manner. Specifically, increasing concentrations of FGF2 inhibits hepatocyte differentiation, whereas intermediate concentration of FGF2 promotes differentiation towards a pancreatic cell fate. At high FGF2 levels specification of midgut endoderm into small intestinal progenitors is increased at the expense of PDX1+ pancreatic progenitors. High FGF2 concentrations also promote differentiation towards an anterior foregut pulmonary cell fate. Finally, by dissecting the FGF receptor intracellular pathway that regulates pancreas specification, we demonstrate for the first time to our knowledge that induction of PDX1+ pancreatic progenitors relies on FGF2-mediated activation of the MAPK signaling pathway. Altogether, these observations suggest a broader gut endodermal patterning activity of FGF2 that corresponds to what has previously been advocated for FGF4, implying a functional switch from FGF4 to FGF2 during evolution. Thus, our results provide new knowledge of how cell fate specification of human DE is controlled - facts that will be of great value for future regenerative cell therapies.
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| 6. |
- Anderson, JD, et al.
(författare)
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Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling
- 2016
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 34:3, s. 601-613
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.
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| 7. |
- Avaliani, Natalia, et al.
(författare)
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Optogenetics reveal delayed afferent synaptogenesis on grafted human induced pluripotent stem cell-derived neural progenitors.
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:12, s. 3088-3098
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Tidskriftsartikel (refereegranskat)abstract
- Reprogramming of somatic cells into pluripotency stem cell state have opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSC), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for six weeks, lt-NES cell-derived neurons displayed neuronal properties such as TTX-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the It-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time-point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, e.g. such as epilepsy. Stem Cells 2014.
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| 8. |
- Azim, Kasum, et al.
(författare)
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Persistent Wnt/β‐Catenin Signaling Determines Dorsalization of the Postnatal Subventricular Zone and Neural Stem Cell Specification into Oligodendrocytes and Glutamatergic Neurons
- 2014
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Ingår i: Stem Cells. - Durham, United States : AlphaMed Press, Inc.. - 1066-5099 .- 1549-4918. ; 32:5, s. 1301-1312
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Tidskriftsartikel (refereegranskat)abstract
- In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/β-catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/β-catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/β-catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/β-catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the β-catenin-activated Wnt-reporter mouse and by pharmacological stimulation of Wnt/β-catenin by infusion of the specific glycogen synthase kinase 3β inhibitor, AR-A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/β-catenin, respectively, increased and decreased the differentiation of dSVZ-NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/β-catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/β-catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/β-catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs.
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| 9. |
- Baris, OR, et al.
(författare)
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The mitochondrial electron transport chain is dispensable for proliferation and differentiation of epidermal progenitor cells
- 2011
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 29:9, s. 1459-1468
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Tidskriftsartikel (refereegranskat)abstract
- Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (TfamEKO) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.
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| 10. |
- Behnan, Jinan, et al.
(författare)
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Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:5, s. 1110-1123
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Tidskriftsartikel (refereegranskat)abstract
- The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These Brain Tumor derived Mesenchymal Stem Cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro, and that the non-MSC population is non-tumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild type GL261 inoculated into GFP-transgenic mice and GL261-GFP cells inoculated into wild type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile, and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. Stem Cells 2013.
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| 11. |
- Berglin-Enquist, Ida, et al.
(författare)
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Successful Low-Risk Hematopoietic Cell Therapy in a Mouse Model of Type 1 Gaucher Disease
- 2009
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:3, s. 744-752
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Tidskriftsartikel (refereegranskat)abstract
- Hematopoietic stem cell-based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, optimization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. In this study we have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a low-risk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of the type 1 GD mouse. Our results demonstrate that a median wild-type (WT) cell engraftment of 7%, corresponding to GCase activity levels above 10 nmoles/hour and mg protein, was sufficient to reverse pathology in bone marrow and spleen in the GD mouse. Moreover, we applied nonmyeloablative doses of busulfan as a pretransplant conditioning regimen and show that even WT cell engraftment in the range of 1%-10% can confer a beneficial therapeutical outcome in this disease model. Taken together, our data provide encouraging evidence for the possibility of developing safe and efficient conditioning protocols for diseases that require only a low level of normal or gene-corrected cells for a permanent and beneficial therapeutic outcome. STEM CELLS 2009; 27: 744-752
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| 12. |
- Bergsland, M, et al.
(författare)
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Nitric oxide-induced neuronal to glial lineage fate-change depends on NRSF/REST function in neural progenitor cells
- 2014
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:9, s. 2539-2549
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Tidskriftsartikel (refereegranskat)abstract
- Degeneration of central nervous system tissue commonly occurs during neuroinflammatory conditions, such as multiple sclerosis and neurotrauma. During such conditions, neural stem/progenitor cell (NPC) populations have been suggested to provide new cells to degenerated areas. In the normal brain, NPCs from the subventricular zone generate neurons that settle in the olfactory bulb or striatum. However, during neuroinflammatory conditions NPCs migrate toward the site of injury to form oligodendrocytes and astrocytes, whereas newly formed neurons are less abundant. Thus, the specific NPC lineage fate decisions appear to respond to signals from the local environment. The instructive signals from inflammation have been suggested to rely on excessive levels of the free radical nitric oxide (NO), which is an essential component of the innate immune response, as NO promotes neuronal to glial cell fate conversion of differentiating rat NPCs in vitro. Here, we demonstrate that the NO-induced neuronal to glial fate conversion is dependent on the transcription factor neuron-restrictive silencing factor-1 (NRSF)/repressor element-1 silencing transcription (REST). Chromatin modification status of a number of neuronal and glial lineage restricted genes was altered upon NO-exposure. These changes coincided with gene expression alterations, demonstrating a global shift toward glial potential. Interestingly, by blocking the function of NRSF/REST, alterations in chromatin modifications were lost and the NO-induced neuronal to glial switch was suppressed. This implicates NRSF/REST as a key factor in the NPC-specific response to innate immunity and suggests a novel mechanism by which signaling from inflamed tissue promotes the formation of glial cells. Stem Cells 2014;32:2539–2549
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| 13. |
- Bigdeli, Narmin, 1974, et al.
(författare)
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Coculture of human embryonic stem cells and human articular chondrocytes results in significantly altered phenotype and improved chondrogenic differentiation.
- 2009
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:8, s. 1812-21
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem (hES) cells have been suggested as a cell source for the repair of cartilage lesions. Here we studied how coculture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers, and differentiation abilities of hES cells, with special regard to chondrogenic differentiation. Undifferentiated hES cells were cocultured with irradiated neonatal or adult articular chondrocytes in high-density pellet mass cultures for 14 days. Cocultured hES cells were then expanded on plastic and their differentiation potential toward the adipogenic, osteogenic, and chondrogenic lineages was compared with that of undifferentiated hES cells. The expression of different surface markers was investigated using flow cytometry and teratoma formation was studied using injection of the cells under the kidney capsule. Our results demonstrate that although hES cells have to be grown on Matrigel, the cocultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Coculture further resulted in a more homogenous pellet and significantly increased cartilage matrix production, both in high-density pellet mass cultures and hyaluronan-based scaffolds. Moreover, cocultured cells formed colonies in agarose suspension culture, also demonstrating differentiation toward chondroprogenitor cells, whereas no colonies were detected in the hES cell cultures. Coculture further resulted in a significantly decreased osteogenic potential. No teratoma formation was detected. Our results confirm the potential of the culture microenvironment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings.
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| 14. |
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| 15. |
- Blanchart, A, et al.
(författare)
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UHRF1 Licensed Self-Renewal of Active Adult Neural Stem Cells
- 2018
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 36:11, s. 1736-1751
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Tidskriftsartikel (refereegranskat)abstract
- Adult neurogenesis in the brain continuously seeds new neurons throughout life, but how homeostasis of adult neural stem cells (NSCs) is maintained is incompletely understood. Here, we demonstrate that the DNA methylation adapter ubiquitin-like, containing PHD and RING finger domains-1 (UHRF1) is expressed in, and regulates proliferation of, the active but not quiescent pool of adult neural progenitor cells. Mice with a neural stem cell-specific deficiency in UHRF1 exhibit a massive depletion of neurogenesis resulting in a collapse of formation of new neurons. In the absence of UHRF1, NSCs unexpectedly remain in the cell cycle but with a 17-fold increased cell cycle length due to a failure of replication phase entry caused by promoter demethylation and derepression of Cdkn1a, which encodes the cyclin-dependent kinase inhibitor p21. UHRF1 does not affect the proportion progenitor cells active within the cell cycle but among these cells, UHRF1 is critical for licensing replication re-entry. Therefore, this study shows that a UHRF1-Cdkn1a axis is essential for the control of stem cell self-renewal and neurogenesis in the adult brain.
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| 16. |
- Blank Savukinas, Ulrika, et al.
(författare)
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The Bystander Effect : Mesenchymal Stem Cell-Mediated Lung Repair
- 2016
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 34:6, s. 1437-1444
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Forskningsöversikt (refereegranskat)abstract
- Mesenchymal stem or stromal cells (MSCs), a heterogeneous subset of adult stem/progenitor cells, have surfaced as potential therapeutic units with significant clinical benefit for a wide spectrum of disease conditions, including those affecting the lung. Although MSCs carry both self-renewal and multilineage differentiation abilities, current dogma holds that MSCs mainly contribute to tissue regeneration and repair by modulating the host tissue via secreted cues. Thus, the therapeutic benefit of MSCs is thought to derive from so called bystander effects. The regenerative mechanisms employed by MSCs in the lung include modulation of the immune system as well as promotion of epithelial and endothelial repair. Apart from secreted factors, a number of recent findings suggest that MSCs engage in mitochondrial transfer and shedding of membrane vesicles as a means to enhance tissue repair following injury. Furthermore, it is becoming increasingly clear that MSCs are an integral component of epithelial lung stem cell niches. As such, MSCs play an important role in coupling information from the environment to stem and progenitor populations, such that homeostasis can be ensured even in the face of injury. It is the aim of this review to outline the major mechanisms by which MSCs contribute to lung regeneration, synthesizing recent preclinical findings with data from clinical trials and potential for future therapy
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| 17. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats
- 2002
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 20:2, s. 163-173
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Tidskriftsartikel (refereegranskat)abstract
- The potential use of in vitro-expanded precursor cells or cell lines in repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide "self-repair." Recently, we have reported that cells from the brain-derived cell line RN33B (derived from the embryonic rat medullary raphe and immortalized through retroviral transduction of the temperature-sensitive mutant of the simian virus 40 ([SV40] large T-antigen) survive for at least 4 weeks, integrate, and differentiate after subretinal grafting to normal adult rats. Here, we demonstrate that grafts of these cells survive for at least 4 months after subretinal transplantation to adult, normal immunosuppressed rats. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve. In addition, the RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A large fraction of the grafted cells differentiate into glial cells, as shown by double labeling of the reporter genes LacZ or green fluorescent protein, and several glial markers, including oligodendrocytes. However, the cells did not differentiate into retinal neurons, judging from their lack of expression of retinal neuronal phenotypic markers. A significant number of the implanted cells in the host retina were in a proliferative stage, judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. To conclude, the cells survived, integrated, and migrated over long distances within the host. Therefore, our results may be advantageous for future design of therapeutic strategies, since such cells may have the potential of being a source of, for example, growth factor delivery in experimental models of retinal degeneration.
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| 18. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Survival and long distance migration of brain-derived precursor cells transplanted to adult rat retina
- 2004
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Ingår i: Stem Cells. - : AlphaMed Press. - 1549-4918 .- 1066-5099. ; 22:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Neural precursor cells transplanted to adult retina can integrate into the host. This is especially true when the neural precursor rat cell line RN33B is used. This cell line carries the reporter genes LacZ and green fluorescent protein (GFP). In grafted rat eyes, RN33B cells are localized from one eccentricity to the other of the host retina. In the present study, whole-mounted retinas were analyzed to obtain a more appropriate evaluation of the amount of transgene-expressing cells and the migratory capacity of these cells 3 and 8 weeks post-transplantation. Quantification was made of the number of beta-galactosidase- and GFP-expressing cells with a semiautomatized stereological cell counting system. With the same system, delineation of the distribution area of the grafted cells was also performed. At 3 weeks, 68% of the grafted eyes contained marker-expressing cells, whereas at 8 weeks only 35% of the eyes contained such cells. Counting of marker-expressing cells demonstrated a lower number of transgene-expressing cells at 3 weeks compared with 8 weeks post-transplantation. The distribution pattern of marker gene-expressing cells revealed cells occupying up to 21% at 3 weeks and up to 68% at 8 weeks of the entire host retina post-grafting. The precursor cells survived well in the adult retina although the most striking feature of the RN33B cell line was its extraordinary migratory capacity. This capability could be useful if precursor cells are used to deliver necessary genes or gene products that need to be distributed over a large diseased area.
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| 19. |
- Bosman, A, et al.
(författare)
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Perturbations of heart development and function in cardiomyocytes from human embryonic stem cells with trisomy 21
- 2015
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 33:5, s. 1434-1446
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Tidskriftsartikel (refereegranskat)abstract
- Congenital heart defects (CHD) occur in approximately 50% of patients with Down syndrome (DS); the mechanisms for this occurrence however remain unknown. In order to understand how these defects evolve in early development in DS, we focused on the earliest stages of cardiogenesis to ascertain perturbations in development leading to CHD. Using a trisomy 21 (T21) sibling human embryonic stem cell (hESC) model of DS, we show that T21-hESC display many significant differences in expression of genes and cell populations associated with mesodermal, and more notably, secondary heart field (SHF) development, in particular a reduced number of ISL1+ progenitor cells. Furthermore, we provide evidence for two candidate genes located on chromosome 21, ETS2 and ERG, whose overexpression during cardiac commitment likely account for the disruption of SHF development, as revealed by downregulation or overexpression experiments. Additionally, we uncover an abnormal electrophysiological phenotype in functional T21 cardiomyocytes, a result further supported by mRNA expression data acquired using RNA-Seq. These data, in combination, revealed a cardiomyocyte-specific phenotype in T21 cardiomyocytes, likely due to the overexpression of genes such as RYR2, NCX, and L-type Ca2+ channel. These results contribute to the understanding of the mechanisms involved in the development of CHD. Stem Cells 2015;33:1434–1446
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| 20. |
- Bottcher, M, et al.
(författare)
-
Mesenchymal Stromal Cells Disrupt mTOR-Signaling and Aerobic Glycolysis During T-Cell Activation
- 2016
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 34:2, s. 516-521
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSCs) possess numerous regenerative and immune modulating functions. Transplantation across histocompatibility barriers is feasible due to their hypo-immunogenicity. MSCs have emerged as promising tools for treating graft-versus-host disease following allogeneic stem cell transplantation. It is well established that their clinical efficacy is substantially attributed to fine-tuning of T-cell responses. At the same time, increasing evidence suggests that metabolic processes control T-cell function and fate. Here, we investigated the MSCs' impact on the metabolic framework of activated T-cells. In fact, MSCs led to mitigated mTOR signaling. This phenomenon was accompanied by a weaker glycolytic response (including glucose uptake, glycolytic rate, and upregulation of glycolytic machinery) toward T-cell activating stimuli. Notably, MSCs express indoleamine-2,3-dioxygenase (IDO), which mediates T-cell suppressive tryptophan catabolism. Our observations suggest that IDO-induced tryptophan depletion interferes with a tryptophan-sufficiency signal that promotes cellular mTOR activation. Despite an immediate suppression of T-cell responses, MSCs foster a metabolically quiescent T-cell phenotype characterized by reduced mTOR signaling and glycolysis, increased autophagy, and lower oxidative stress levels. In fact, those features have previously been shown to promote generation of long-lived memory cells and it remains to be elucidated how MSC-induced metabolic effects shape in vivo T-cell immunity.
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| 21. |
- Brederlau, Anke, 1968, et al.
(författare)
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Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation.
- 2006
-
Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 24:6, s. 1433-40
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinson's disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6-OHDA (6-hydroxydopamine)-lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC-derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion-induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.
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| 22. |
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| 23. |
- Bryja, V, et al.
(författare)
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An efficient method for the derivation of mouse embryonic stem cells
- 2006
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 24:4, s. 844-849
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Tidskriftsartikel (refereegranskat)abstract
- Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%–75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.
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| 24. |
- Carreira, Bruno Pereira, et al.
(författare)
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Nitric Oxide Stimulates the Proliferation of Neural Stem Cells Bypassing the Epidermal Growth Factor Receptor
- 2010
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28:7, s. 1219-1230
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Tidskriftsartikel (refereegranskat)abstract
- Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 mu M) increased cell proliferation, whereas higher concentrations (100 mu M) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor I, p27(KIP1), allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS(-/-) mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS 2010:28:1219-1230
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| 25. |
- Charbord, J, et al.
(författare)
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High throughput screening for inhibitors of REST in neural derivatives of human embryonic stem cells reveals a chemical compound that promotes expression of neuronal genes
- 2013
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 31:9, s. 1816-1828
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Tidskriftsartikel (refereegranskat)abstract
- Decreased expression of neuronal genes such as brain-derived neurotrophic factor (BDNF) is associated with several neurological disorders. One molecular mechanism associated with Huntington disease (HD) is a discrete increase in the nuclear activity of the transcriptional repressor REST/NRSF binding to repressor element-1 (RE1) sequences. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to identify two benzoimidazole-5-carboxamide derivatives that inhibited REST silencing in a RE1-dependent manner. The most potent compound, X5050, targeted REST degradation, but neither REST expression, RNA splicing nor binding to RE1 sequence. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST in wild-type neural cells treated with X5050. This activity was confirmed in neural cells produced from human induced pluripotent stem cells derived from a HD patient. Acute intraventricular delivery of X5050 increased the expressions of BDNF and several other REST-regulated genes in the prefrontal cortex of mice with quinolinate-induced striatal lesions. This study demonstrates that the use of pluripotent stem cell derivatives can represent a crucial step toward the identification of pharmacological compounds with therapeutic potential in neurological affections involving decreased expression of neuronal genes associated to increased REST activity, such as Huntington disease.
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| 26. |
- Costa, FF, et al.
(författare)
-
Concise review: cancer/testis antigens, stem cells, and cancer
- 2007
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 25:3, s. 707-711
-
Tidskriftsartikel (refereegranskat)abstract
- In the multistep process of cancer development, the concept that cancer stem cells are derived from normal stem cells that have gradually accumulated various genetic and epigenetic defects is gaining strong evidence. A number of investigations have identified molecular markers that, under normal conditions, are responsible for stem cell homeostasis but are also expressed in tumor “stem cell-like” subpopulations. In this regard, it was recently reported that a group of tumor-specific antigens known as cancer/testis antigens (CTAs) are expressed in human MSCs. It has long been stated that in normal tissue these antigens are exclusively expressed in germ cell precursors; however, based on these results, we suggest that CTAs are expressed at earlier stages during embryogenesis. The tumor-restricted expression of CTAs has led to several immunotherapeutic trials targeting some of these proteins. The clinical implications that these trials may have on the normal stem cell pools, as well as the immunologic properties of these cells, is to date poorly studied and should be considered.
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| 27. |
- Covacu, Ruxandra, et al.
(författare)
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Nitric oxide exposure diverts neural stem cell fate from neurogenesis towards astrogliogenesis
- 2006
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 178, s. 268-268
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Regeneration of cells in the central nervous system is a process that might be affected during neurological disease and trauma. Because nitric oxide (NO) and its derivatives are powerful mediators in the inflammatory cascade, we have investigated the effects of pathophysiological concentrations of NO on neurogenesis, gliogenesis, and the expression of proneural genes in primary adult neural stem cell cultures. After exposure to NO, neurogenesis was downregulated, and this corresponded to decreased expression of the proneural gene neurogenin-2 and beta-III-tubulin. The decreased ability to generate neurons was also found to be transmitted to the progeny of the cells. NO exposure was instead beneficial for astroglial differentiation, which was confirmed by increased activation of the Janus tyrosine kinase/signal transducer and activator of transcription transduction pathway. Our findings reveal a new role for NO during neuroinflammatory conditions, whereby its proastroglial fate-determining effect on neural stem cells might directly influence the neuroregenerative process.
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| 28. |
- Cusulin, Carlo, et al.
(författare)
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Embryonic Stem Cell-Derived Neural Stem Cells Fuse with Microglia and Mature Neurons.
- 2012
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099.
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation of neural stem cells (NSCs) is a novel strategy to restore function in the diseased brain, acting through multiple mechanisms, e.g., neuronal replacement, neuroprotection and modulation of inflammation. Whether transplanted NSCs can operate by fusing with microglial cells or mature neurons is largely unknown. Here we have studied the interaction of a mouse embryonic stem cell-derived neural stem (NS) cell line with rat and mouse microglia and neurons in vitro and in vivo. We show that NS cells spontaneously fuse with co-cultured cortical neurons, and that this process requires the presence of microglia. Our in vitro data indicate that the NS cells can first fuse with microglia, and then with neurons. The fused NS/microglial cells express markers and retain genetic and functional characteristics of both parental cell types, being able to respond to microglia-specific stimuli (LPS and IL-4/IL-13) and to differentiate to neurons and astrocytes. The NS cells fuse with microglia, at least partly, through interaction between phosphatidylserine (PS) exposed on the surface of NS cells and CD36 receptor on microglia. Transplantation of NS cells into rodent cortex results in fusion with mature pyramidal neurons, which often carry two nuclei, a process probably mediated by microglia. The fusogenic role of microglia could be even more important after NSC transplantation into brains affected by neurodegenerative diseases associated with microglia activation. It remains to be elucidated how the occurrence of the fused cells will influence the functional outcome after NSC transplantation in the diseased brain.
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| 29. |
- Davies, LC, et al.
(författare)
-
Mesenchymal Stromal Cell Secretion of Programmed Death-1 Ligands Regulates T Cell Mediated Immunosuppression
- 2017
-
Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 35:3, s. 766-776
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells of innate and adaptive immune systems. As MSCs become accepted as a therapeutic option for the treatment of immunological disorders such as Graft versus Host Disease, our need to understand the intricate details by which they exert their effects is crucial. Programmed death-1 (PD-1) is an important regulator in T cell activation and homeostatic control. It has been reported that this pathway may be important in contact-dependent mediated immunomodulation by MSCs. The aim of this study was to establish whether MSCs, in addition to their cell-surface expression, are able to secrete PD-1 ligands (PD-L1 and PD-L2) and their potential importance in modulating contact-independent mechanisms of MSC immunosuppression. Here we report that MSCs express and secrete PD-L1 and PD-L2 and that this is regulated by exposure to interferon γ and tumor necrosis factor α. MSCs, via their secretion of PD-1 ligands, suppress the activation of CD4+ T cells, downregulate interleukin-2 secretion and induce irreversible hyporesponsiveness and cell death. Suppressed T cells demonstrated a reduction in AKT phosphorylation at T308 and a subsequent increase in FOXO3 expression that could be reversed with blockade of PD-L1. In conclusion, we demonstrate for the first time, that MSCs are able to secrete PD-1 ligands, with this being the first known report of a biological role for PD-L2 in MSCs. These soluble factors play an important role in modulating immunosuppressive effects of MSCs directly on T cell behavior and induction of peripheral tolerance.
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| 30. |
- Delsing, Louise, et al.
(författare)
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Barrier properties and transcriptome expression in human iPSC-derived models of the blood-brain barrier
- 2018
-
Ingår i: Stem Cells. - : AlphaMed Press, Inc.. - 1066-5099 .- 1549-4918. ; 36:12, s. 1816-1827
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Tidskriftsartikel (refereegranskat)abstract
- Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of inter-species differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in co-culture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our co-culture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in co-culture, including upregulation of tight junction proteins such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF and PI3K-Akt pathways upon co-culture. Our data suggests that co-culture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in co-culture can be used to further improve iPSC-derived BBB models through selective pathway manipulation.
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| 31. |
- Denham, Mark, et al.
(författare)
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Gli1 Is an Inducing Factor in Generating Floor Plate Progenitor Cells from Human Embryonic Stem Cells
- 2010
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28:10, s. 1805-1815
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Tidskriftsartikel (refereegranskat)abstract
- Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer. STEM CELLS 2010; 28: 1805-1815
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| 32. |
- Draganova, Kalina, et al.
(författare)
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Wnt/β‐Catenin Signaling Regulates Sequential Fate Decisions of Murine Cortical Precursor Cells
- 2015
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Ingår i: Stem Cells. - Durham, United States : AlphaMed Press, Inc.. - 1066-5099 .- 1549-4918. ; 33:1, s. 170-182
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Tidskriftsartikel (refereegranskat)abstract
- The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. β-Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of β-catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel β-catenin mutant allele with conditional inactivation approaches. Wnt/β-catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/β-catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper-layer neurons and later, to precocious astrogenesis. Indeed, a genome-wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/β-catenin signaling-ablated cortex. Thus, β-catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage-specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain.
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| 33. |
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| 34. |
- Ellerström, Catharina, et al.
(författare)
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Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation
- 2007
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 25:7, s. 1690-1696
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Tidskriftsartikel (refereegranskat)abstract
- Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations.
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| 35. |
- Fritze, Jonas, et al.
(författare)
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Loss of Cxcr5 alters neuroblast proliferation and migration in the aged brain
- 2020
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 38:9, s. 1175-1187
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Tidskriftsartikel (refereegranskat)abstract
- Neurogenesis, the production of new neurons from neural stem cells, dramatically decreases during aging concomitantly with increased inflammation both systemically and in the brain. However, the precise role of inflammation and whether local or systemic factors drive the neurogenic decline during aging is poorly understood. Here, we identify CXCR5/5/CXCL13 signaling as a novel regulator of neurogenesis in the aged brain. The chemokine Cxcl13 was found to be upregulated in the brain during aging. Loss of its receptor, Cxcr5, led to increased proliferation and decreased numbers of neuroblasts in the aged subventricular zone (SVZ), together with accumulation of neuroblasts in the rostral migratory stream and olfactory bulb (OB), without increasing the amount of new mature neurons in the OB. The effect on proliferation and migration was specific to neuroblasts and likely mediated through increased levels of systemic IL-6 and local Cxcl12 expression in the SVZ. Our study raises the possibility of a new mechanism by which interplay between systemic and local alterations in inflammation regulates neurogenesis during aging.
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| 36. |
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| 37. |
- Gao, H, et al.
(författare)
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CD36 Is a Marker of Human Adipocyte Progenitors with Pronounced Adipogenic and Triglyceride Accumulation Potential
- 2017
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 35:7, s. 1799-1814
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Tidskriftsartikel (refereegranskat)abstract
- White adipose tissue (WAT) expands in part through adipogenesis, a process involving fat cell generation and fatty acid (FA) storage into triglycerides (TGs). Several findings suggest that inter-individual and regional variations in adipogenesis are linked to metabolic complications. We aimed to identify cellular markers that define human adipocyte progenitors (APs) with pronounced adipogenic/TG storage ability. Using an unbiased single cell screen of passaged human adipose-derived stromal cells (hADSCs), we identified cell clones with similar proliferation rates but discordant capabilities to undergo adipogenic differentiation. Transcriptomic analyses prior to induction of differentiation showed that adipogenic clones displayed a significantly higher expression of CD36, encoding the scavenger receptor CD36. CD36+ hADSCs, in comparison with CD36-cells, displayed almost complete adipogenic differentiation while CD36 RNAi attenuated lipid accumulation. Similar findings were observed in primary CD45-/CD34+/CD31-APs isolated from human WAT where the subpopulation of MSCA1+/CD36+ cells displayed a significantly higher differentiation degree/TG storage capacity than MSCA1+/CD36-cells. Functional analyses in vitro and ex vivo confirmed that CD36 conferred APs an increased capacity to take up FAs thereby facilitating terminal differentiation. Among primary APs from subcutaneous femoral, abdominal and visceral human WAT, the fraction of CD36+ cells was significantly higher in depots associated with higher adipogenesis and reduced metabolic risk (i.e., femoral WAT). We conclude that CD36 marks APs with pronounced adipogenic potential, most probably by facilitating lipid uptake. This may be of value in developing human adipocyte cell clones and possibly in linking regional variations in adipogenesis to metabolic phenotype.
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| 38. |
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| 39. |
- Genander, M, et al.
(författare)
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Ephrins negatively regulate cell proliferation in the epidermis and hair follicle
- 2010
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28:7, s. 1196-1205
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Tidskriftsartikel (refereegranskat)abstract
- Ephrins and their Eph tyrosine kinase receptors control many processes during embryonic development. They have more recently also been identified as important regulators of proliferation of stem/progenitor cells in the adult brain and intestine and have been implicated in tumorigenesis in a large number of tissues. We here describe the expression of a large number of ephrins and Eph receptors in the adult mouse skin. Disruption of the ephrin-Eph interaction in vivo with antagonists against the A or B class resulted in an approximate doubling of cell proliferation in the hair follicle and epidermis of adult mice. We conclude that ephrins are negative regulators of proliferation in the skin and that blocking the ephrin-Eph interaction may be an attractive strategy for regenerative therapies.
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| 40. |
- Grinnemo, Karl-Henrik, et al.
(författare)
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Costimulation blockade induces tolerance to HESC transplanted to the testis and induces regulatory T-cells to HESC transplanted into the heart
- 2008
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 26:7, s. 1850-1857
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Tidskriftsartikel (refereegranskat)abstract
- In order to study the ability of costimulation blockade to induce tolerance to human embryonic stem cells (HESC), severe combined immunodeficient (SCID), and immunocompetent C57BL/6 mice treated with costimulation blockade received intratesticular and intramyocardial HESC transplants. All SCID mice with intratesticular HESC transplants developed teratoma. When SCID mice were transplanted intramyocardially, only two of five mice developed teratoma-like tumors. C57BL/6 mice transplanted intratesticularly and treated with costimulation blockade all developed teratoma and were surrounded by CD4(+)CD25(+)Foxp3(+) T-cells, while isotype control treated recipients rejected their grafts. Most C57BL/6 mice transplanted intramyocardially and treated with costimulation blockade demonstrated lymphocytic infiltrates 1 month after transplantation, whereas one maintained its graft. Isolation of regulatory T-cells from intramyocardial transplanted recipients treated with costimulation blockade demonstrated specificity toward undifferentiated HESC and down-regulated naive T-cell activation toward HESC. These results demonstrate that costimulation blockade is sufficiently robust to induce tolerance to HESC in the immune-privileged environment of the testis. HESC specific regulatory T-cells developed to HESC transplanted to the heart and the success of transplantation was similar to that seen in SCID mice.
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| 41. |
- Hall, Vanessa, et al.
(författare)
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Using therapeutic cloning to fight human disease: A conundrum or reality?
- 2006
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 24:7, s. 1628-1637
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Tidskriftsartikel (refereegranskat)abstract
- The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.
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| 42. |
- Hansson, Mats G., et al.
(författare)
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Commentary: Isolated Stem Cells - Patentable as Cultural Artifacts?
- 2007
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 25:6, s. 1507-1510
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Tidskriftsartikel (refereegranskat)abstract
- This article argues that an isolated embryonic stem cell basically represents a cultural artifact that has no equivalent to cells of the embryo, and that it is likely that the isolation of adult stem cells has a similar consequence. An isolated stem cell could thus be distinguished as something other than the stem cell existing as part of a human body. Since isolation of stem cells implies modification, product patents should, where the results carry enough novelty, inventive step, and potential for industrial application, as a matter of principle be a viable option for patent authorities. Questions of morality, which may affect the patentability, should also be viewed in light of the distinction between isolated result and body part. At the same time, it is essential that patent authorities do not accept broad patent claims that will be detrimental to research. Disclosure of potential conflicts of interest is found at the end of this article.
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| 43. |
- Hara, Y, et al.
(författare)
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Impaired hippocampal neurogenesis and vascular formation in ephrin-A5-deficient mice
- 2010
-
Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28:5, s. 974-983
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Tidskriftsartikel (refereegranskat)abstract
- Neurogenesis occurs throughout the life in the mammalian brain. The hippocampal dentate gyrus (DG) is one of the major regions of the adult neurogenesis, where neural stem/progenitor cells continuously generate new granule neurons, although molecular mechanisms underlying generation and maintenance of newly born neurons are still elusive. Here we show that ephrin-A5, a ligand for Eph receptor tyrosine kinases, plays multiple roles in both neurogenesis and vascular formation in the adult hippocampus. In mice lacking ephrin-A5 function, cell proliferation and survival of newborn neurons were severely reduced in the hippocampus DG. Furthermore, ephrin-A5-deficient mice exhibited altered distribution of EphA4 receptor in the vascular endothelial cells and increased narrower capillaries in the hippocampus DG. EphA/ephrin-A signaling thus plays crucial roles in the establishment and/or maintenance of the brain vascular system, as an essential constituent of the adult neurogenic niche.
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| 44. |
- Heins, Nico, et al.
(författare)
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Derivation, characterization, and differentiation of human embryonic stem cells.
- 2004
-
Ingår i: Stem cells (Dayton, Ohio). - 1066-5099. ; 22:3, s. 367-76
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Tidskriftsartikel (refereegranskat)abstract
- The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.
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| 45. |
- Heiskanen, Annamari, et al.
(författare)
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N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible.
- 2007
-
Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 25:1, s. 197-202
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.
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| 46. |
- Heldin, Carl-Henrik
(författare)
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Signal transduction : multiple pathways, multiple options for therapy
- 2001
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 19:4, s. 295-303
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Tidskriftsartikel (refereegranskat)abstract
- Many aspects of cell behavior, such as growth, motility, differentiation, and apoptosis, are regulated by signals cells receive from their environment. Such signals are important, e.g., during embryonal development, wound healing, hematopoiesis, and in the regulation of the immune response, and may come from interactions with other cells or components of the extracellular matrix, or from binding of soluble signaling molecules to specific receptors at the cell membrane. Hereby different signaling pathways are initiated inside the cell. Perturbations of such signaling pathways are seen in several types of diseases, e.g., cancer, inflammatory conditions, and atherosclerosis. Thus, antagonists of several signaling pathways have potential clinical utility. Several such compounds are currently used or are in clinical trials; others are currently being analyzed in animal models.
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| 47. |
- Hellström, Nina, 1976, et al.
(författare)
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Differential recovery of neural stem cells in the subventricular zone and dentate gyrus after ionizing radiation.
- 2009
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:3, s. 634-641
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Tidskriftsartikel (refereegranskat)abstract
- Radiation therapy is a widely used treatment for malignant CNS tumors. Mature neurons are terminally differentiated, whereas stem and progenitor cells have a prominent proliferative capacity and are therefore highly vulnerable to irradiation. Our aim was to investigate how cranial radiation in young rats would affect stem/progenitor cells in the two niches of adult neurogenesis, the subventricular zone (SVZ) and the dentate gyrus of the hippocampal formation. Nine weeks after irradiation we found that in irradiated animals, hippocampal neurogenesis was reduced to 5% of control levels. Similarly, the number of actively proliferating cells and radial glia-like stem cells (nestin+/GFAP+) in the dentate gyrus, was reduced to 10% and 15% of control levels, respectively. In the irradiated olfactory bulb, neurogenesis was reduced to 40% of control levels and the number of actively proliferating cells in the SVZ was reduced to 53% of control levels. However, the number of nestin+/GFAP+ cells in the SVZ was unchanged compared to controls. To evaluate the immediate response to the radiation injury we quantified the amount of proliferation in the SVZ and dentate gyrus one day after irradiation. We found an equal reduction in proliferating cells both in dentate gyrus and SVZ. In summary, we show an initial response to radiation injury that is similar in both brain stem cell niches. However, the long-term effects on stem cells and neurogenesis in these two areas differ significantly, where the dentate gyrus is severely affected long-term, whereas the SVZ appears to recover with time. ______________________________________________________________________________
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| 48. |
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| 49. |
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| 50. |
- Jeon, Iksoo, et al.
(författare)
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Neuronal Properties, In Vivo Effects, and Pathology of a Huntington's Disease Patient-Derived Induced Pluripotent Stem Cells
- 2012
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 30:9, s. 2054-2062
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Tidskriftsartikel (refereegranskat)abstract
- Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can be used to model different human diseases. They may also serve as sources of transplantable cells that can be used in novel cell therapies. Here, we analyzed neuronal properties of an iPSC line derived from a patient with a juvenile form of Huntington's disease (HD) carrying 72 CAG repeats (HD-iPSC). Although its initial neural inducing activity was lower than that of human embryonic stem cells, we found that HD-iPSC can give rise to GABAergic striatal neurons, the neuronal cell type that is most susceptible to degeneration in HD. We then transplanted HD-iPSC-derived neural precursors into a rat model of HD with a unilateral excitotoxic striatal lesion and observed a significant behavioral recovery in the grafted rats. Interestingly, during our in vitro culture and when the grafts were examined at 12 weeks after transplantation, no aggregate formation was detected. However, when the culture was treated with a proteasome inhibitor (MG132) or when the cells engrafted into neonatal brains were analyzed at 33 weeks, there were clear signs of HD pathology. Taken together, these results indicate that, although HD-iPSC carrying 72 CAG repeats can form GABAergic neurons and give rise to functional effects in vivo, without showing an overt HD phenotype, it is highly susceptible to proteasome inhibition and develops HD pathology at later stages of transplantation. These unique features of HD-iPSC will serve as useful tools to study HD pathology and develop novel therapeutics. Stem Cells 2012; 30: 20542062
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