Cortical Stem Cells Research Articles

Abstract 554: Mechanism of Action for the Beneficial Effects of Cortical Bone Stem Cells on the Heart After Myocardial Infarction

Rationale: Myocardial infarction (MI) causes the death of a portion of the heart and can lead to adverse remodeling that results in poor cardiac pump function and heart failure. We have previously shown that a novel stem cell type, cortical bone stem cells (CBSCs), causes improved repair of cardiac tissue after MI. When injected into the infarct border zone in both mouse and pig hearts, CBSCs reduced infarct size and induced neovascularization via a largely paracrine manner. Objectives: This study investigates the mechanism involved in the angiogenesis alterations implemented by CBSCs, in particular the wound-healing of endothelial cells. Our goal was to comprehend how mCBSCs affected cardiac repair in an inflammatory environment. Methods and Results: We performed scratch assays and tubule formation (TF) assays on human umbilical vein endothelial cells (HUVECs) and cardiac endothelial cells (CECs) in mCBSC CM and media controls, and in the presence of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. Both HUVECs and CECs show reduced cell migration and reduced TF. TF was rescued when the cells were plated on Matrigel. We investigated the angiogenesis-related cytokines present within both the mCBSC CM and CM-treated HUVECs and CECs via angiogenesis dot blots. We discovered a significant increase of CXCL-10 and Endostatin/Collagen XVIII relative to murine embryonic fibroblast CM. Conclusions: Our findings show that part of the mechanism underlying the wound-healing and neovascularization induced by CBSCs post-MI involves secretion of angiogenesis-related cytokines which alter the behavior of cardiac endothelial cells.

Application of a Demineralized Cortical Bone Matrix and Peripheral Blood-Derived Mesenchymal Stem Cells in A Rabbit Total Meniscectomy Model

Application of a Demineralized Cortical Bone Matrix and Peripheral Blood-Derived Mesenchymal Stem Cells in A Rabbit Total Meniscectomy Model

The role of the Eph/ephrin family during cortical development and cerebral malformations

Neuronal numbers and the associated size of the cerebral cortex, surface folding and laminar organisation are determined by precise developmental mechanisms that are orchestrated by several intrinsic and extrinsic molecules. Abnormalities during development can cause manifold microscopic and macroscopic cortical malformations, mostly accompanied by clinical consequences such as mental disorders, intellectual disabilities, or epileptic seizures. Most cortical malformations and associated neurological disorders result from genetic defects, however the cellular mechanisms remain complex and poorly understood. Eph receptor tyrosine kinases and their ligands, the ephrins, are abundantly expressed in the developing brain where they regulate several developmental processes that are crucial for correct brain formation. Ephrin family members represent membrane-bound proteins that are key players in complex short-range cell-cell communication. In addition, mechanisms for long-range interactions have been described recently. Several ephrins have already been shown to control cell cycle dynamics of cortical stem cells during corticogenesis and the positioning of postmitotic neurons. In addition, mutations in genes encoding for members of the Eph/ephrin family are implicated in mental disorders, although the underlying mechanisms remain to be elucidated. A deeper understanding of Eph/ephrin interactions during cerebral cortex development will be beneficial to shed light on developmental disabilities. Here, we discuss the function of Eph/ephrin system during the different processes of corticogenesis and the impact on cerebral malformations.

Absence of Carboxypeptidase E/Neurotrophic Factor-Α1 in Knock-Out Mice Leads to Dysfunction of BDNF-TRKB Signaling in Hippocampus.

Carboxypeptidase E (CPE), first discovered as a prohormone processing enzyme, has also now been shown to be a secreted neurotrophic factor (neurotrophic factor-α1, NF-α1) that acts extracellularly as a signaling molecule to mediate neuroprotection, cortical stem cell differentiation, and antidepressive-like behavior in mice. Since brain-derived neurotrophic factor (BDNF) has very similar trophic functions, and its processing from pro-BDNF involves intracellular sorting of pro-BDNF to the regulated secretory pathway by CPE acting as a sorting receptor, we investigated whether the lack of CPE/NF-α1 would affect BDNF-TrkB signaling in mice. Previous studies have shown that CPE/NF-α1 knock-out (KO) mice exhibited severe neurodegeneration of the hippocampal CA3 region which raises the question of why other neurotrophic factors such as BDNF could not compensate for the deficiency of CPE. Here, we show that the expressions of pro-BDNF mRNA and protein in hippocampus of CPE-KO mice were similar to WT mice, but mature BDNF was ∼40% less in the CPE-KO mice, suggesting decreased intracellular processing of pro-BDNF. Furthermore, TrkB receptor levels were similar in both genotypes, but there was significantly decreased phosphorylation of TrkB receptor in the CPE-KO mice. Electrophysiological studies showed lack of formation of long-term potentiation in hippocampal slices of CPE-KO mice compared to WT mice, which was not rescued by application of BDNF, indicating dysfunction of the BDNF-TrkB signaling system. The CPE-KO mice showed normal postsynaptic AMPA response to kainate application in hippocampal slices and dissociated neurons. Our findings indicate that CPE/NF-α1 is essential for normal BDNF-TrkB signaling function in mouse hippocampus.

Extracellular Regulation of the Mitotic Spindle and Fate Determinants Driving Asymmetric Cell Division.

Stem cells use mode of cell division, symmetric (SCD) versus asymmetric (ACD), to balance expansion with self-renewal and the generation of daughter cells with different cell fates. Studies in model organisms have identified intrinsic mechanisms that govern this process, which involves partitioning molecular components between daughter cells, frequently through the regulation of the mitotic spindle. Research performed in vertebrate tissues is revealing both conservation of these intrinsic mechanisms and crucial roles for extrinsic cues in regulating the frequency of these divisions. Morphogens and positional cues, including planar cell polarity proteins and guidance molecules, regulate key signaling pathways required to organize cell/ECM contacts and spindle pole dynamics. Noncanonical WNT7A/VANGL2 signaling governs asymmetric cell division and the acquisition of cell fates through spindle pole orientation in satellite stem cells of regenerating muscle fibers. During cortical neurogenesis, the same pathway regulates glial cell fate determination by regulating spindle size, independent of its orientation. Sonic hedgehog (SHH) stimulates the symmetric expansion of cortical stem and cerebellar progenitor cells and contributes to cell fate acquisition in collaboration with Notch and Wnt signaling pathways. SLIT2 also contributes to stem cell homeostasis by restricting ACD frequency through the regulation of spindle orientation. The capacity to influence stem cells makes these secreted factors excellent targets for therapeutic strategies designed to enhance cell populations in degenerative disease or restrict cell proliferation in different types of cancers.

Neurotrophic Factor-α1: A Key Wnt-β-Catenin Dependent Anti-Proliferation Factor and ERK-Sox9 Activated Inducer of Embryonic Neural Stem Cell Differentiation to Astrocytes in Neurodevelopment.

Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells (NSCs) followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of NSC/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing β-catenin. NF-α1 induced differentiation of NSCs to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and Western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-Knockout mice showed ∼49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating antiproliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment. Stem Cells 2017;35:557-571.

Abstract 21: Effect of Hypoxic Preconditioning on Cortical Bone Stem Cells (CBSCs)

Rationale: Cortical Bone Stem Cells (CBSCs) improve cardiac performance when delivered in an infarcted heart, in part, through secretion of paracrine factors. However transplanted cells can die due to hypoxia or other hostile fators within the infarcted heart. Hypoxia is known to also influence several biological process including migration, proliferation and secretory activity. Objective: the aim of this study is to define the effects of hypoxia on biological properties of CBSCs. Methods and Results: CBSCs were isolated from C57Bl/6 mouse, expanded in vitro and characterized. CBSCs were subjected to hypoxia (1% O 2 ) or normoxia (21% O 2 ) condition and then several biological properties were analysed. Hypoxia resulted in upregulation of CAIX and GLUT1 expression, two HIF1α downstream targets (3.2 and 5.1 fold increase of GLUT1; 6.7 and 65 fold increase of CAIX after 6 and 24 hours respectively). This indicates an effective hypoxic response activation in our experimental conditions. Cell proliferation was assessed by CyQuant assay: no significant difference between normoxic and hypoxic cells were found after 1 and 2 days. Growth curves were determined to evaluate hypoxia effects at longer time points. Hypoxic cells had the same growing rate as cells in normoxia in the first days, but their number increased at longer time points, indicating different kinetics with respect to normoxic when cells were grown in a challenging condition like an exhausted media. CBSCs migration and chemotaxis decreased when cells were cultured at 1%O 2 . Hypoxia preconditioning increased the paracrine activity of CBSCs. VEGF expression was increased after 6 hours (3 fold), and this effect remain stable after 24 hours and 3 days of 1% O 2 . Hypoxia resulted in 2.2 and 1.9 fold increase of SDF1 after 24 hours and 3 days respectively and a 1.8 fold increase of IGF1 after 1 day. The expression of bFGF, Angiopoietin and SCF was not affected. Conclusion: CBSCs can survive hypoxia and their paracrine activity increases without adversely affecting their proliferative capacity. In vivo experiments are needed to investigate if hypoxic preconditioning will enhance the ability of CBSCs to improve the structure and function of the injured heart.

Abstract 364: Cortical Bone Stem Cells Derived Exosomes as Potent Modulator of Cardiac Immune Response and Repair After Injury

Rationale: Cortical bone derived stem cells (CBSCs) are known to have improved growth kinetics and myocardial repair properties that are superior to other known stem cell types used. Salutary effects of CBSCs in large are mediated by paracrine secretion. Since exosomes represent an active component of released factors we tested if CBSC derived exosomes (CBSCs-Exo) can recapitulate the beneficial reparative effects of CBSCs. Objective: Determine CBSCs derived exosomes and their contents for myocardial repair. Methods and Results: Exosomes were isolated from murine CBSCs by ultracentrifugation and had typical size (30-100nm), as validated by electron microscopy and dynamic light scattering. To determine cardiac therapeutic value, CBSCs- Exo (60μg) were injected into the border zone of the mouse heart after myocardial infarction (MI). Animals injected with CBSCs-Exo had reduced infarct size and increased myocyte survival after MI injury. Interestingly, serum levels of pro-inflammatory cytokines were significantly reduced along with decreased expression of CD68+ cells in animals receiving CBSCs-Exo versus control animals. Long term analysis of CBSC-Exo animals showed improved cardiac function and contractility compared to saline treated animals concurrent with enhanced angiogenesis 6 weeks after MI. Salutary effects of CBSC-Exo were confirmed in vitro. CBSCs-Exo increased cardiac protection in NRVMs after hypoxic challenge and enhanced tube formation in HUVECs. Simultaneously, treatment of bone marrow-derived macrophages stimulated with lipopolysaccharide (LPS) and treated with CBSCs-Exo showed increased polarization towards the M2 phenotype, demonstrating an immunomodulatory capacity of CBSCs-Exo. The underlying mechanism for beneficial effects was linked to increased packaging of cardioprotective miRs including miR125, miR20 and miR18a in CBSCs-Exo confirmed by MiRNA array analysis. Conclusion: Exosomes derived from CBSCs provide a cell free system that retains the reparative power of CBSC. CBSCs-Exo augment cardiac function after myocardial injury recapitulating earlier findings with CBSCs. The packaging of cardioprotective and immune-modulatory miRs in CBSCs-Exo appears to enhance their reparative effects after MI.

Abstract 2: Cortical Bone Stem Cells Derived Exosomes as Potent Modulator of Cardiac Immune Response and Repair After Injury

Rationale: Cortical bone derived stem cells (CBSCs) are known to have improved growth kinetics and myocardial repair properties that are superior to other known stem cell types used. Salutary effects of CBSCs in large are mediated by paracrine secretion. Since exosomes represent an active component of released factors we tested if CBSC derived exosomes (CBSCs-Exo) can recapitulate the beneficial reparative effects of CBSCs. Objective: Determine CBSCs derived exosomes and their contents for myocardial repair. Methods and Results: Exosomes were isolated from murine CBSCs by ultracentrifugation and had typical size (30-100nm), as validated by electron microscopy and dynamic light scattering. To determine cardiac therapeutic value, CBSCs- Exo (60μg) were injected into the border zone of the mouse heart after myocardial infarction (MI). Animals injected with CBSCs-Exo had reduced infarct size and increased myocyte survival after MI injury. Interestingly, serum levels of pro-inflammatory cytokines were significantly reduced along with decreased expression of CD68+ cells in animals receiving CBSCs-Exo versus control animals. Long term analysis of CBSC-Exo animals showed improved cardiac function and contractility compared to saline treated animals concurrent with enhanced angiogenesis 6 weeks after MI. Salutary effects of CBSC-Exo were confirmed in vitro. CBSCs-Exo increased cardiac protection in NRVMs after hypoxic challenge and enhanced tube formation in HUVECs. Simultaneously, treatment of bone marrow-derived macrophages stimulated with lipopolysaccharide (LPS) and treated with CBSCs-Exo showed increased polarization towards the M2 phenotype, demonstrating an immunomodulatory capacity of CBSCs-Exo. The underlying mechanism for beneficial effects was linked to increased packaging of cardioprotective miRs including miR125, miR20 and miR18a in CBSCs-Exo confirmed by MiRNA array analysis. Conclusion: Exosomes derived from CBSCs provide a cell free system that retains the reparative power of CBSC. CBSCs-Exo augment cardiac function after myocardial injury recapitulating earlier findings with CBSCs. The packaging of cardioprotective and immune-modulatory miRs in CBSCs-Exo appears to enhance their reparative effects after MI.

Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity.

Mitochondrial dysfunction connects metabolic disturbance with numerous pathologies, but the significance of mitochondrial activity in bone remains unclear. We have, therefore, characterized the skeletal phenotype in the Opa3L122P mouse model for Costeff syndrome, in which a missense mutation of the mitochondrial membrane protein, Opa3, impairs mitochondrial activity resulting in visual and metabolic dysfunction. Although widely expressed in the developing normal mouse head, Opa3 expression was restricted after E14.5 to the retina, brain, teeth and mandibular bone. Opa3 was also expressed in adult tibiae, including at the trabecular surfaces and in cortical osteocytes, epiphyseal chondrocytes, marrow adipocytes and mesenchymal stem cell rosettes. Opa3L122P mice displayed craniofacial abnormalities, including undergrowth of the lower mandible, accompanied in some individuals by cranial asymmetry and incisor malocclusion. Opa3L122P mice showed an 8-fold elevation in tibial marrow adiposity, due largely to increased adipogenesis. In addition, femoral length and cortical diameter and wall thickness were reduced, the weakening of the calcified tissue and the geometric component of strength reducing overall cortical strength in Opa3L122P mice by 65%. In lumbar vertebrae reduced vertebral body area and wall thickness were accompanied by a proportionate reduction in marrow adiposity. Although the total biomechanical strength of lumbar vertebrae was reduced by 35%, the strength of the calcified tissue (σmax) was proportionate to a 38% increase in trabecular number. Thus, mitochondrial function is important for the development and maintenance of skeletal integrity, impaired bone growth and strength, particularly in limb bones, representing a significant new feature of the Costeff syndrome phenotype.

Novel insights into mammalian embryonic neural stem cell division: focus on microtubules.

During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic “rule breakers,” control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals.

Abstract 18293: Unique Features of Cortical Bone Stem Cells Associated With Enhanced Cardiac Repair

Rationale: Adoptive transfer of multiple stem cell types into failing human hearts has demonstrated modest beneficial effects in patients with cardiovascular disorders that damage the heart. Despite increasing use of stem cell based therapies, consensus on the optimal stem cell type is still not clear. Modest repair and improvement in cardiac function in patients with cardiac complications warrants identification of a novel stem cell population that possesses enhanced proliferative capacity and can effectively survives the harsh myocardial environment. Objective: To characterize surface marker expression, proliferation, survival, migration and differentiation capacity of CBSCs relative to known and extensively studied stem cell types including MSCs and CDCs. Methods and Results: CBSCs, MSCs and CDCs were isolated from Gottingen miniswine. CBSCs possess a unique cell surface marker profile versus CDCs and MSCs. Additionally CBSCs were morphologically distinct from MSCs and CDCs and showed enhanced proliferation capacity versus CDCs and MSCs. Concurrently CBSCs had significantly increased percentage of survival after exposure to an apoptotic stimuli as compared to MSC. A significantly greater percentage of CBSCs expressed markers of cardiac lineage commitment. Additionally, CBSCs can form functional gap junction with adult ventricular myocytes. CBSCs, CDCs and MSCs showed differential response towards ATP and histamine further strengthening the differences between the three cell types. Conclusion: CBSCs have enhanced proliferative capacity and cardiac lineage commitment versus CDCs and MSCs that could account for their enhanced effects on cardiac regeneration after myocardial infarction. Ultimately, this understanding would help us to develop viable cellular therapy options for treatment of heart failure patients.

Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects.

BackgroundIn recent years, we have come to recognize that a multitude of in utero exposures have the capacity to induce the development of congenital and metabolic defects. As most of these encounters manifest their effects beyond the window of exposure, deciphering the mechanisms of teratogenesis is incredibly difficult. For many agents, altered epigenetic programming has become suspect in transmitting the lasting signature of exposure leading to dysgenesis. However, while several chemicals can perturb chromatin structure acutely, for many agents (particularly alcohol) it remains unclear if these modifications represent transient responses to exposure or heritable lesions leading to pathology.ResultsHere, we report that mice encountering an acute exposure to alcohol on gestational Day-7 exhibit significant alterations in chromatin structure (histone 3 lysine 9 dimethylation, lysine 9 acetylation, and lysine 27 trimethylation) at Day-17, and that these changes strongly correlate with the development of craniofacial and central nervous system defects. Using a neural cortical stem cell model, we find that the epigenetic changes arising as a consequence of alcohol exposure are heavily dependent on the gene under investigation, the dose of alcohol encountered, and that the signatures arising acutely differ significantly from those observed after a 4-day recovery period. Importantly, the changes observed post-recovery are consistent with those modeled in vivo, and associate with alterations in transcripts encoding multiple homeobox genes directing neurogenesis. Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription. Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1.ConclusionsThese observations suggest that the immediate and long-term impacts of alcohol exposure on chromatin structure are distinct, and hint at the existence of a possible coordinated epigenetic response to ethanol during development. Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0031-7) contains supplementary material, which is available to authorized users.

Abstract 314: Allogeneic Cortical Bone Stem Cells Preserve Cardiac Structure and Function by Inhibition of Cell Death and Enhancing Angiogenesis in the Swine Heart After Myocardial Infarction

Introduction: Novel therapies are needed to improve cardiac function after MI; one strategy is to replace lost myocardium. Despite the success of bone marrow- and cardiac- stem cell clinical trials, we’re still searching for the optimal stem cell type most suitable for cardiac regeneration. Previously, we described a novel cell population derived from the cortical bone (CBSCs) which repaired the heart post MI via transdifferentiation and paracrine signaling mechanisms in a mouse model. In the present study, we evaluate the translational potential of allogeneic CBSCs in swine MI model. Hypothesis: Intramyocardial injection of CBSCs into the MI border zone immediately after reperfusion will preserve cardiac structure and pump function by enhancing endogenous repair through secretion of paracrine factors. Methods & Results: Female Göttingen minipigs received MI via occlusion of the left anterior descending coronary artery (LAD) for 90 minutes, followed by reperfusion. Animals received either 20 million CBSCs (via 10 intramyocardial injections) or saline injections. Cardiac structure and function was evaluated using echocardiography at baseline and 4 weeks post-MI. During the 4 weeks after MI there was depression if pump function in saline treated group (EF: 69.27% ± 1.92 [baseline] to 46.305% ± 3.53 [4 weeks post MI] p<0.0001 ) and dilation of the LV (EDV: 39.01 ml ± 3.47 [baseline] to 51.5 ml ± 5.67 [4 weeks post MI] and ESV was 13.71 ml ± 2.47 to 27.72 ml ± 3.86 ( p= 0.0136 ). The MI + CBSC group had no significant change in ventricular pump function from baseline to 4 weeks post-MI; while demonstrating preservation of cardiac structure indicating a decrease in ventricular remodeling/scar formation. A decrease in TUNEL+ cells and an increase in angiogenesis was also observed in CBSC’s treated animals. Conclusion: CBSC’s appear to reduce infarct expansion by reducing post MI myocyte death and by increasing angiogenesis. These data show that administration of CBSCs immediately after reperfusion of an infarcted region of the heart can reduce adverse cardiac remodeling and improve cardiac function.

Abstract 157: Unique Features of Cortical Bone Stem Cells Associated With Enhanced Cardiac Repair

Rationale: Adoptive transfer of bone marrow and cardiac derived stem cells (CDCs) into failing human hearts has been shown to be safe, yet these cells have only induced modest improvements after myocardial infarction (MI). Recently we have shown in a mouse model that cortical bone derived stem cells (CBSCs) induced a greater enhancement of cardiac function after MI through enhanced paracrine signaling and transdifferentiation of CBSCs into new cardiac tissue. However, the reparative potential of CBSCs relative to other stem cell types including bone marrow derived mesenchymal stem cells (MSCs) and CDCs is not known. Objective: To characterize surface marker expression, proliferation, survival, migration and differentiation capacity of swine CBSCs relative to MSCs and CDCs. Methods and Results: CBSCs, MSCs and CDCs were isolated from Gottingen miniswine. CBSCs were morphologically distinct from MSCs and CDCs, with differences in length to width ratio and overall cell surface area. Cell surface marker profiling using RT-PCR analysis revealed that CBSCs express some of the classical MSC markers such as CD106, CD271, CD105, CD90 and CD29 and are negative for CD45 and CD11-b. CBSCs had an enhanced proliferation capacity versus CDCs and MSCs, measured by CyQuant assay. Concurrently CBSCs had significantly decreased population-doubling time (3.57 and 1.26 fold decrease) as compared to MSC and CDCs. CBSCs exhibit enhanced survival after exposure to apoptotic stimuli as compared to MSCs measured by Annexin-V staining. A significantly greater % of CBSCs expressed markers of cardiac lineage commitment when exposed to dexamethasone than did CDCs or MSCs. Markers of cardiac lineages including GATA-4, α SMA, Troponin T, sm22 were measured with RT-PCR and immunocytochemistry. Conclusion: CBSCs have enhanced proliferative, survival capacity and cardiac lineage commitment versus CDCs and MSCs that could account for their enhanced effects on cardiac regeneration after myocardial infarction.

Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPK(Ser-485), but not AMPK(Thr-172), phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPK(Ser-485), but not AMPK(Thr-172), hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPK(Ser-485) hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPK(S485A) mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPK(Ser-485) hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPK(Ser-485) phosphorylation to the increased risk of AD in obesity and diabetes.

DYRK1A-mediated Cyclin D1 Degradation in Neural Stem Cells Contributes to the Neurogenic Cortical Defects in Down Syndrome

Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.

Abstract 17423: Unique Features of Cortical Bone Stem Cells Associated With Enhanced Cardiac Repair

Rationale: Adoptive transfer of bone marrow and cardiac derived stem cells (CDCs) into failing human hearts has been shown to be safe, yet these cells have induced modest improvements in myocardial function. Recently we have shown in a mouse model that cortical bone derived stem cells (CBSCs) induced a greater enhancement of cardiac function after myocardial infarction than CDCs, possibly through enhanced paracrine signaling and transdifferentiation of the transplanted cells. However, the relative reparative potential of CBSCs relative to other known stem cell types including bone marrow derived mesenchymal stem cells (MSCs) and cardiac derived stem cells (CDCs) is not known. Objective: To characterize surface marker expression, proliferation, survival and differentiation capacity of swine CBSCs relative to MSCs and CDCs. Methods and Results: CBSCs, MSCs and CDCs were isolated from cortical bone, bone marrow and heart (left ventricle) of Gottingen miniswine. CBSCs were morphologically distinct from MSCs and CDCs, with differences in length to width ratio and overall cell surface area. Cell surface marker profiling using RT-PCR analysis revealed that CBSCs express some of the classical MSC markers such as CD106, CD271, CD105, CD90, CD44 and CD29 and are negative for CD45 and CD11-b. CBSCs had an enhanced proliferation capacity versus CPCs and MSCs, measured by CyQuant assay. Concurrently CBSCs had significantly a decreased population doubling time (3.57 and 1.26 fold decrease) as compared to MSC and CDCs respectively. CBSCs were also more hydrogen peroxide stress tolerant than CSCs and MSCs as measured by Annexin-V and propidium iodide (PI) labeling using flow cytometery. A significantly greater % of CBSCs expressed markers of cardiac lineage commitment when exposed to dexamethasone than did CSCs or MSCs. Markers of cardiac lineages including GATA-4, α SMA, Troponin T, Nkx2.5, sm22 were measured with RT-PCR and immunocytochemistry. Conclusion: CBSCs have enhanced proliferative capacity, survival in oxidative stress conditions and cardiac lineage commitment versus CSCs and MSCs. These features make them a promising cell source for cardiac regeneration after myocardial infarction.

The complexity of the calretinin-expressing progenitors in the human cerebral cortex.

The complex structure and function of the cerebral cortex critically depend on the balance of excitation and inhibition provided by the pyramidal projection neurons and GABAergic interneurons, respectively. The calretinin-expressing (CalR+) cell is a subtype of GABAergic cortical interneurons that is more prevalent in humans than in rodents. In rodents, CalR+ interneurons originate in the caudal ganglionic eminence (CGE) from Gsx2+ progenitors, but in humans it has been suggested that a subpopulation of CalR+ cells can also be generated in the cortical ventricular/subventricular zone (VZ/SVZ). The progenitors for cortically generated CalR+ subpopulation in primates are not yet characterized. Hence, the aim of this study was to identify patterns of expression of the transcription factors (TFs) that commit cortical stem cells to the CalR fate, with a focus on Gsx2. First, we studied the expression of Gsx2 and its downstream effectors, Ascl1 and Sp8 in the cortical regions of the fetal human forebrain at midgestation. Next, we established that a subpopulation of cells expressing these TFs are proliferating in the cortical SVZ, and can be co-labeled with CalR. The presence and proliferation of Gsx2+ cells, not only in the ventral telencephalon (GE) as previously reported, but also in the cerebral cortex suggests cortical origin of a subpopulation of CalR+ neurons in humans. In vitro treatment of human cortical progenitors with Sonic hedgehog (Shh), an important morphogen in the specification of interneurons, decreased levels of Ascl1 and Sp8 proteins, but did not affect Gsx2 levels. Taken together, our ex-vivo and in vitro results on human fetal brain suggest complex endogenous and exogenous regulation of TFs implied in the specification of different subtypes of CalR+ cortical interneurons.

Gabapentin Enhances Neurogenesis in E14 Rat Embryonic Neocortex Stem Cells.

Many anticonvulsant drugs have been studied for their non conventional therapeutic effects on neurodegerative diseases but merely a few demonstrated potential neurogenic characteristic. Gabapentin as a well-known mood stabilizer was studied for its potential capability to promote neurogenesis in embryonic rat cortical stem cells. Rat E14 cortical stem cells were exposed to gabapentin during differentiation for 7 days and subjected to immunocytochemistry. The phenotypic changes were evaluated in the ultimately survived and differentiated cells. Gabapentin (16 µg/ml) exposure significantly increased the number and percentage of MAP2 immunopositive neurons with no significant alterations in nestin or GFAP immunopositivity in neural or glial progenitors. The enhanced number of neurons by therapeutic doses of gabapentin via augmentation of the neuronal differentiation in neural stem cells may participate to the therapeutic properties of gabapentin in the treatment of mood disorder.