CNS Stem Cells Research Articles

Cell Replacement Therapy for Retinal and Optic Nerve Diseases: Cell Sources, Clinical Trials and Challenges.

The aim of this review was to provide an update on the potential of cell therapies to restore or replace damaged and/or lost cells in retinal degenerative and optic nerve diseases, describing the available cell sources and the challenges involved in such treatments when these techniques are applied in real clinical practice. Sources include human fetal retinal stem cells, allogenic cadaveric human cells, adult hippocampal neural stem cells, human CNS stem cells, ciliary pigmented epithelial cells, limbal stem cells, retinal progenitor cells (RPCs), human pluripotent stem cells (PSCs) (including both human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs)) and mesenchymal stem cells (MSCs). Of these, RPCs, PSCs and MSCs have already entered early-stage clinical trials since they can all differentiate into RPE, photoreceptors or ganglion cells, and have demonstrated safety, while showing some indicators of efficacy. Stem/progenitor cell therapies for retinal diseases still have some drawbacks, such as the inhibition of proliferation and/or differentiation in vitro (with the exception of RPE) and the limited long-term survival and functioning of grafts in vivo. Some other issues remain to be solved concerning the clinical translation of cell-based therapy, including (1) the ability to enrich for specific retinal subtypes; (2) cell survival; (3) cell delivery, which may need to incorporate a scaffold to induce correct cell polarization, which increases the size of the retinotomy in surgery and, therefore, the chance of severe complications; (4) the need to induce a localized retinal detachment to perform the subretinal placement of the transplanted cell; (5) the evaluation of the risk of tumor formation caused by the undifferentiated stem cells and prolific progenitor cells. Despite these challenges, stem/progenitor cells represent the most promising strategy for retinal and optic nerve disease treatment in the near future, and therapeutics assisted by gene techniques, neuroprotective compounds and artificial devices can be applied to fulfil clinical needs.

SP-009 - In vitro evaluation of GPR17 radioligands as potential CNS stem cell imaging agents

SP-009 - In vitro evaluation of GPR17 radioligands as potential CNS stem cell imaging agents

Abstract SY39-03: Stem cell competition is central to leukemogenesis

Abstract Tissue stem cells regenerate the body and are the only cells that maintain self-renewal throughout life. When stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by differentiation). Stem cell isolation and transplantation is the basis for regenerative medicine. We isolated mouse and then human hematopoietic stem cells (HSCs). Importantly, we demonstrated that transplantation of purified HSCs results in complete regeneration of the blood and immune systems without causing graft vs. host disease, and can induce permanent transplant tolerance of any organ or cell from the HSC donor. In a clinical trial in metastatic breast cancer patients, HSC purification made autologous transplantation to cancer patients possible, without including cancerous cells in the graft. The 15-year overall survival rate was ~7% in recipients of mobilized peripheral blood (MPB) autologous transplantation, and ~33% in recipients of cancer-free HSCs. We have also prospectively isolated human fetal brain CNS stem cells and found a method to propagate them without changing phenotype or normal regenerative functions upon transplantation. These are and have been used in clinical trials with children with genetic neurodegenerative diseases such as Batten and Pelizeus Mehrbacher, in patients with thoracic and cervical spinal cord injuries, and in patients with age-related macular degeneration. Self-renewal is strictly regulated and restricted to stem cells, because deregulation can lead to cancer. To study the relationship between stem cells and cancer, we followed the progression from hematopoietic stem cells (HSCs) to myelogenous leukemias. We found that the developing precancerous clones progress and accumulate mutations at the stage of HSCs, until they become fully malignant. At this point, the “leukemia” stem cell is a downstream oligolineage or multilineage progenitor that has evaded programmed cell death and programmed cell removal, and also acquired self-renewal. In the case of chronic myeloid leukemia (CML), bcr-abl+ HSC clones outcompete normal HSCs in the chronic phase. The transition from CML to myeloid blast crisis results in the leukemia stem cells appearing in the granulocyte-macrophage progenitor (GMP) stage, and is accompanied by cell intrinsic activation of β-catenin, endowing the already mutant progenitors with a self-renewal capacity. While there are many ways to defeat programmed cell death and senescence, there appears to be one dominant method that cancer cells use to avoid programmed cell removal—the elevated expression of the cell surface “don't eat me” protein CD47, the ligand for macrophage SIRPα. All cancers tested express CD47 to overcome expression of “eat me” signals such as calreticulin that is produced by macrophages and adheres to asialogylycoproteins on the surface of cancer cells. Antibodies that block the CD47-SIRPα interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo. This includes human solid tumors and lymphomas/leukemias/myelomas. We showed that anti-CD47 antibodies or high-affinity SIRPα proteins synergize with anti-CD20 antibodies to eliminate human non-Hodgkin lymphoma in immune-deficient mice. We generated a humanized antibody of the IgG4 isotype, and these compounds are currently being tested in clinical trials. Citation Format: Irving L. Weissman. Stem cell competition is central to leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY39-03.

OS01.1 Identification of the pre-malignant cell of origin in Human Glioblastoma Multiforme

AbstractOne of the major difficulties in identifying the cell of origin for glioblastoma is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. To identify the cell of origin in human GBM we decided to first dissect out the various stages of CNS stem cell differentiation in fetal and adult human brain. After a functional high throughput screen, we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90) that in various combinations can separate long-term self-renewing multi-potent neural stem cells (NSCs: LinCD15Notch1EGFRCD90) from multi-potent progenitors with limited self-renewal capacity (NPC: LinCD15Notch1EGFRCD90) from non-neoplastic human fetal and adult Brain (sub-ventricular zone). FACS sorted cells were analyzed for single cell lineage multi-potentiality, lineage bias and self-renewal to trace and characterize their lineage relationships. In surgical human glioblastoma samples, however we observed in-vivo tumor initiating and in-vitro tumorsphere-forming frequency was predominant in the progenitor cells and not in the neural stem cells. Interestingly we did observe a small frequency of NSC cells forming distinct lesions in the brain, which on further analysis showed the presence of both the neural stem cells and Glioma stem cells, where as the tumor initiating enriched population (GSC: LinCD15Notch1EGFRCD90) did not show the presence of neural stem cells but only tumor initiating NPCs. limiting dilution transplantation and serial transplantation confirmed the LinCD15Notch1EGFRCD90 to have the highest frequency of self renewing cells compared to other populations. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. Using Transcriptome analysis and targeted sequencing of known mutations we identify the signal transduction and genomic events (PTEN point mutations vs Deletions) which distinguish pre-malignant neural stem cells from the tumor initiating glioma stem cells. We further show a significant enrichment of the protein translational machinery as cells transition from pre-malignant to tumor initiating cells. These pre-malignant cells comprise a cellular reservoir that needs to be targeted to prevent tumor relapse. SSM: Seibel Fellowship and Ludwig Cancer Center, ILW: National Institutes of Health, Ludwig Cancer Center, SHC: Stanford Child Health Research Institute, St Baldrick’s Foundation and Ludwig Cancer Center

Recruitment of endogenous CNS stem cells for regeneration in demyelinating disease.

Demyelinating diseases, such as multiple sclerosis (MS), are responsible for a significant portion of the neurological disability burden worldwide, especially in young adults. Demyelination can be followed by a spontaneous regenerative process called remyelination, in which new myelin sheaths are restored to denuded axons. However, in chronic demyelinating disease such as MS, this process becomes progressively less efficient. This chapter reviews the biology of remyelination and the rationale and strategies by which it can be enhanced therapeutically in acquired demyelinating disease.

Hedgehog Controls Quiescence and Activation of Neural Stem Cells in the Adult Ventricular-Subventricular Zone.

SummaryIdentifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.

Distinct transcription factor complexes act on a permissive chromatin landscape to establish regionalized gene expression in CNS stem cells.

Spatially distinct gene expression profiles in neural stem cells (NSCs) are a prerequisite to the formation of neuronal diversity, but how these arise from the regulatory interactions between chromatin accessibility and transcription factor activity has remained unclear. Here, we demonstrate that, despite their distinct gene expression profiles, NSCs of the mouse cortex and spinal cord share the majority of their DNase I hypersensitive sites (DHSs). Regardless of this similarity, domain-specific gene expression is highly correlated with the relative accessibility of associated DHSs, as determined by sequence read density. Notably, the binding pattern of the general NSC transcription factor SOX2 is also largely cell type specific and coincides with an enrichment of LHX2 motifs in the cortex and HOXA9 motifs in the spinal cord. Interestingly, in a zebrafish reporter gene system, these motifs were critical determinants of patterned gene expression along the rostral-caudal axis. Our findings establish a predictive model for patterned NSC gene expression, whereby domain-specific expression of LHX2 and HOX proteins act on their target motifs within commonly accessible cis-regulatory regions to specify SOX2 binding. In turn, this binding correlates strongly with these DHSs relative accessibility—a robust predictor of neighboring gene expression.

Abstract A11: Identification of the premalignant cell of origin in human glioblastoma multiforme

Abstract One of the major difficulties in identifying the cell of origin for glioblastoma is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. To identify the human cell of origin we delineated the stages of CNS stem cell differentiation in fetal and adult human brain. After a functional high throughput screen, we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90) that in various combinations can separate long-term self-renewing multi-potent neural stem cells (NSCs) from multi-potent progenitors with limited self-renewal capacity (NPC) from non-neoplastic human fetal and adult Brain (sub-ventricular zone). FACS sorted cells were analyzed for single cell lineage multi-potentiality, lineage bias and self-renewal to trace and characterize their lineage relationships. In surgical human glioblastoma samples, however we observed in-vivo tumor initiating and in-vitro tumorsphere-forming frequency was predominant in the progenitor cells and not in the neural stem cells. Interestingly we did observe a small frequency of NSC cells forming distinct lesions in the brain, which on further analysis showed the presence of both the NSC and NPC, whereas the tumor initiating enriched population did not show the presence of the NSC but only tumor initiating NPCs. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, RNAseq and targeted sequencing of known mutations identified the genomic events which can distinguish these pre-malignant neural stem cells from the tumor initiating cancer stem cells. These pre-malignant cells could comprise a cellular reservoir that may need to be targeted to prevent tumor relapse. Citation Format: Siddhartha S. Mitra, Sharareh Gholamin, Rahul Sinha, Rachel M. Morganti, Irving Weissman, Samuel H. Cheshier. Identification of the premalignant cell of origin in human glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A11.

WNT signaling in microglia and the glioma microenvironment.

WNT signaling in microglia and the glioma microenvironment Gunnar Schulte WNT signaling is important during embryonic development and organogenesis having specific roles in the development of the CNS such as regulation of neural tube formation, axon guidance and CNS stem cell regulation. Our work has recently established a role of WNT signaling in the regulation of the brain’s macrophages, the microglia and thus WNTs emerge as novel regulators of CNS inflammatory responses. First of all, it appeared that b-catenin levels are elevated in microglia in Alzheimer disease (AD) brains as well as microglia cells in AD mouse models. Employing in vitro studies of primary mouse microglia isolated from newborn mouse pups indicated that both WNT-3A and WNT-5A induce diverse signaling routes in microglia leading to differential proinflammatory modulation of the cells. Interestingly, the net effect of WNT stimulation on the inflammatory potential of mouse microglia is context dependent. While WNTs increase inflammatory markers when giving to microglia alone, they are able to act in an anti-inflammatory manner when microglia are activated by prestimulation with lipopolysaccharides. Our findings thereby indicate that WNTs act on microglia as a homeostatic regulator, further underlined by yet unpublished data that suggest that WNT-5A is elevated in human glioma associated with a distinct inflammatory signature of the tumor as well as a substantial invasion of microglia.

PM-15 * C11orf95-RELA FUSION ALONE IS SUFFICIENT TO FORM HUMAN SUPRATENTORIAL EPENDYMOMA-LIKE TUMOR IN MICE

Ependymomas (EMs) are primary tumors of the central nervous system that preferentially occur in children. Recent deep molecular analyses have revealed that these tumors are divided into molecularly distinct subgroups; particularly supra- and infratentorial EMs have significantly different genetic alterations. Chromothripsis is a massive genomic rearrangement event rarely occurring in various types of tumors, potentially resulting in oncogenic gene fusion. Recently, several types of C11orf95-associated fusion gene caused by gene rearrangements via chromothripsis were identified in a large fraction of supratentorial EMs. These fusion genes have been shown to be oncogenic when transferred to CNS stem cells in allograft model, these fusion genes may be promising therapeutic targets for the tumor type. Pysiologically relevant genetic mouse models are useful for the understanding of oncogenic mechanisms and drivers and for the exploration of theraputic targets in particular tumor types. Therefore, we used the RCAS/tv-a system to determine the ability for the C11orf95-RELA fusion to induce the formation of ependymomas in vivo in mice. When RCAS vector expressing the C11orf95-RELA fusion was introduced into Nestin/tv-a mice, tumors formed in a few months. Additional Cdkn2a loss in the context leads to an increased tumor penetrance. These tumors were observed as a circumscribed tumor adjacent to ventricular surface and formed abundant branching vascular network in the tumor, likely recapitulating histologic features of the human counterpart. This model should be a useful tool for understanding EM biology, therapeutic response, and recurrence.

From CNS stem cells to neurons and glia: Sox for everyone.

Neuroepithelial precursor cells of the vertebrate central nervous system either self-renew or differentiate into neurons, oligodendrocytes or astrocytes under the influence of a gene regulatory network that consists in transcription factors, epigenetic modifiers and microRNAs. Sox transcription factors are central to this regulatory network, especially members of the SoxB, SoxC, SoxD, SoxE and SoxF groups. These Sox proteins are widely expressed in neuroepithelial precursor cells and in newly specified, differentiating and mature neurons, oligodendrocytes and astrocytes and influence their identity, survival and development. They exert their effect predominantly at the transcriptional level but also have substantial impact on expression at the epigenetic and posttranscriptional levels with some Sox proteins acting as pioneer factors, recruiting chromatin-modifying and -remodelling complexes or influencing microRNA expression. They interact with a large variety of other transcription factors and influence the expression of regulatory molecules and effector genes in a cell-type-specific and temporally controlled manner. As versatile regulators with context-dependent functions, they are not only indispensable for central nervous system development but might also be instrumental for the development of reprogramming and cell conversion strategies for replacement therapies and for assisted regeneration after injury or degeneration-induced cell loss in the central nervous system.

Injury to the Spinal Cord Niche Alters the Engraftment Dynamics of Human Neural Stem Cells

SummaryThe microenvironment is a critical mediator of stem cell survival, proliferation, migration, and differentiation. The majority of preclinical studies involving transplantation of neural stem cells (NSCs) into the CNS have focused on injured or degenerating microenvironments, leaving a dearth of information as to how NSCs differentially respond to intact versus damaged CNS. Furthermore, single, terminal histological endpoints predominate, providing limited insight into the spatiotemporal dynamics of NSC engraftment and migration. We investigated the early and long-term engraftment dynamics of human CNS stem cells propagated as neurospheres (hCNS-SCns) following transplantation into uninjured versus subacutely injured spinal cords of immunodeficient NOD-scid mice. We stereologically quantified engraftment, survival, proliferation, migration, and differentiation at 1, 7, 14, 28, and 98 days posttransplantation, and identified injury-dependent alterations. Notably, the injured microenvironment decreased hCNS-SCns survival, delayed and altered the location of proliferation, influenced both total and fate-specific migration, and promoted oligodendrocyte maturation.

196 Human Neural Stem Cell Transplantation in Chronic SCI

INTRODUCTION: Demonstration of the safety of human neural stem cells is critical for the overall development of intramedullary transplantation following spinal cord injury (SCI). We present interim results of an on-going Phase I/II trial 12 months after transplantation of human CNS stem cells (HuCNS-SC®, StemCells, Inc., Newark, California) in patients with chronic thoracic (T2-11) SCI. Special emphasis will be put on aspects of surgical administration and methods of thoracic segmental assessment. We will also address preliminary clinical neurophysiological and radiological outcomes following stem cell transplantation. METHODS: Three ASIA Impairment Scale (AIS) A paraplegic patients were transplanted with allogenic HuCNS-SC cells between 3-12 months post-injury. RESULTS: There were no surgical complications or adverse changes directly attributed to the cells at the 12 months follow-up. Signs of segmental improvement were observed in 2 out of 3 AIS A patients, and the third subject remained stable. Based on the safety results in the first AIS A patients, recruitment of patients with incomplete injury (AIS B and C patients) has now commenced. CONCLUSION: We present 12 months interim safety and preliminary efficacy data of the first 3 AIS A SCI patients who underwent thoracic intramedullary transplantation of HuCNS-SC as part of an on-going open-label Phase I/II trial.

Abstract 5007: Delineating human fetal CNS stem cell hierarchy reveals a progenitor cell of origin for human Gliobastoma Multiforme.

Abstract Glioblastoma Multiforme (GBM) is the most common and highly aggressive human brain tumor. Even with great advancement of surgical procedures and treatment regimens with radiation and chemotherapy, malignant gliomas remain incurable. This is due to their resistance to all conventional therapies and the diffuse infiltrative nature of the tumor cells, which makes complete tumor resection impossible. One of the major difficulties for the identification of the cell of origin for gliomas is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. In order to identify the cell of origin we decided to first dissect out the various stages of CNS stem cell differentiation. After testing a panel of 34 antibodies to prospectively isolate neurospheres forming cells from human fetal brain tissue we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90). Subpopulations of cells derived after FACS were analyzed for single cell lineage potency and the ability to generate neurospheres. Using an in-vitro limiting dilution assay, we ascertained that double-positive (DP; CD15+Notch1+EGFR-CD90-)) populations have the highest frequency of self-renewing cells, followed by quadruple-positive (QP) populations (CD15+Notch1+EGFR-CD90-). In-vitro differentiation assays reveal both DP and QP to be multipotent in nature however serial passaging showed that only the DP cells have a consistent self-renewing capacity, whereas QP derived cells were unable to survive after 5-6 passages. In glioma samples however we observed by limiting dilution and serial passaging assays that the tumorsphere forming frequency was predominant in the QP population and not in the DP populations. Furthermore the GBM derived DP cells serially passaged if grown under normal neural stem cell growth condition suggesting that this population is still at a pre-malignant state. In-vivo tumorigenic assay via orthotopic implation of as few as a 1000 cells into mouse brain showed significantly higher tumor formation from the QP cells and not in the DP cells. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. These pre-malignant cells could comprise a cellular reservoir that may need to be targeted for future therapies. Citation Format: Siddhartha S. Mitra, Sharareh Gholamin, Abdullah Feroze, Samuel H. Cheshier, Irving L. Weissman. Delineating human fetal CNS stem cell hierarchy reveals a progenitor cell of origin for human Gliobastoma Multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2013-5007

Central nervous system stem cell transplantation for children with neuronal ceroid lipofuscinosis

Infantile and late-infantile neuronal ceroid lipofuscinoses (NCLs) are invariably fatal lysosomal storage diseases associated with defects in lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT-1) or tripeptidyl peptidase 1 (TPP1) activity. Previous preclinical studies have demonstrated that human CNS stem cells (HuCNS-SCs) produce both PPT-1 and TPP1 and result in donor cell engraftment and reduced accumulation of storage material in the brain when tested in an NCL mouse model. HuCNS-SC transplantation was tested in an open-label dose-escalation Phase I clinical trial as a potential treatment for infantile and late-infantile NCL. Study design included direct neurosurgical transplantation of allogeneic HuCNS-SCs into the cerebral hemispheres and lateral ventricles accompanied by 12 months of immunosuppression. Six children with either the infantile or late-infantile forms of NCL underwent low- (3 patients) and high- (3 patients) dose transplantation of HuCNS-SCs followed by immunosuppression. The surgery, immunosuppression, and cell transplantation were well tolerated. Adverse events following transplantation were consistent with the underlying disease, and none were directly attributed to the donor cells. Observations regarding efficacy of the intervention were limited by the enrollment criteria requiring that patients be in advanced stages of disease. This study represents the first-in-human clinical trial involving transplantation of a purified population of human neural stem cells for a neurodegenerative disorder. The feasibility of this approach and absence of transplantation-related serious adverse events support further exploration of HuCNS-SC transplantation as a potential treatment for select subtypes of NCL, and possibly for other neurodegenerative disorders.

Sox21 Promotes Hippocampal Adult Neurogenesis via the Transcriptional Repression of theHes5Gene

Despite the importance of the production of new neurons in the adult hippocampus, the transcription network governing this process remains poorly understood. The High Mobility Group (HMG)-box transcription factor, Sox2, and the cell surface activated transcriptional regulator, Notch, play important roles in CNS stem cells. Here, we demonstrate that another member of the SoxB (Sox1/Sox2/Sox3) transcription factor family, Sox21, is also a critical regulator of adult neurogenesis in mouse hippocampus. Loss of Sox21 impaired transition of progenitor cells from type 2a to type 2b, thereby reducing subsequent production of new neurons in the adult dentate gyrus. Analysis of the Sox21 binding sites in neural stem/progenitor cells indicated that the Notch-responsive gene, Hes5, was a target of Sox21. Sox21 repressed Hes5 gene expression at the transcriptional level. Simultaneous overexpression of Hes5 and Sox21 revealed that Hes5 was a downstream effector of Sox21 at the point where the Notch and Sox pathways intersect to control the number of neurons in the adult hippocampus. Therefore, Sox21 controls hippocampal adult neurogenesis via transcriptional repression of the Hes5 gene.

The synergistic effect of beta-boswellic acid and Nurr1 overexpression on dopaminergic programming of antioxidant glutathione peroxidase-1-expressing murine embryonic stem cells

Parkinson’s disease (PD) is a neurodegenerative disorder in which the nigro-striatal dopaminergic (DAergic) neurons have been selectively lost. Due to side effects of levodopa, a dopamine precursor drug, recently cell replacement therapy for PD has been considered. Lack of sufficient amounts of, embryos and ethical problems regarding the use of dopamine-rich embryonic neural cells have limited the application of these cells for PD cell therapy. Therefore, many investigators have focused on using the pluripotent stem cells to generate DAergic neurons. This study is aimed first to establish a mouse embryonic stem (mES) cell line that can stably co-express Nurr1 (Nuclear receptor subfamily 4, group A, member 2) transcription factor in order to efficiently generate DAergic neurons, and glutathione peroxidase-1 (GPX-1) to protect the differentiated DAergic-like cells against oxidative stress. In addition to genetic engineering of ES cells, the effect of Beta-boswellic acid (BBA) on DAergic differentiation course of mES cells was sought in the present study. To that end, the feeder-independent CGR8 mouse embryonic stem cells were transduced by Nurr1- and GPX-1-harboring Lentiviruses and the generated Nurr1/GPX-1-expresssing ES clones were characterized and verified. Gene expression analyses demonstrated that BBA treatment and overexpression of Nurr1 has a synergistic effect on derivation of DAergic neurons from Nurr1/GPX-1-expressing ES cells. The differentiated cells could exclusively synthesize and secrete dopamine in response to stimuli. Overexpression of GPX-1 in genetically engineered Nurr1/GPX-1-ES cells increased the viability of these cells during their differentiation into CNS stem cells. In conclusion, the results demonstrated that Nurr1-overexpressing feeder-independent ES cells like the feeder-dependent ES cells, can be efficiently programmed into functional DAergic neurons and additional treatment of cells by BBA can even augment this efficiency. GPX-1 overexpression in Nurr1/GPX-1-ES cells increases the viability of differentiated CNS stem-like cells. The result of this study may have impact on future stem cell therapy of PD.

Re-Engineering the Subthalamus

This historical vignette of the region known as “subthalamus” is timely and brings up how our current concepts regarding this region have evolved into the current level of high clinical importance for clinical treatment schemes. This pertinent review of older literature (particularly prior to 1990) highlights how previous concepts as to functioning and treatment in this region have been redefined and improved (1). Whereas most efforts at treatment of this region are now focused on motor disorders (ie, tremor and Parkinson’s disease) this vignette outlines the wide variety of other disorders once considered as involving the subthalamus, including epilepsy (16), chronic pain, and behavioral disorders, as neurosurgeons experimented to define the function of this region. Additionally, the authors include previous treatment paradigms (ie, various forms of lesioning, the previous mainstay of generating a treatment effect), now mostly supplanted by long-term electrical stimulation with deep brain stimulation (DBS), gene therapy approaches to modify long-term cellular function, and other biological or cellular treatments, all focused on augmentation of function rather than lesioning.

Abstract 1372: Microvessel proliferation by co-expression of endothelial nestin and Ki-67 is associated with a basal-like phenotype and aggressive features in breast cancer

Abstract Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential in tumour growth and metastasis. Microvessel density (MVD) is the most widely used method for quantification of tumour angiogenesis. We here present a novel angiogenesis marker, microvessel proliferation (MVP) based on a dual immunohistochemical stain of nestin and Ki-67. Nestin is an intermediate filament protein expressed in a variety of undifferentiated cells, including newly synthesized endothelial cells (1-3), and used in this study to stain the vasculature. Proliferating cells were recognized by their Ki-67 positivity, and immature microvessels were recognized by their nestin positivity combined with their morphology. Proliferating microvessels contains endothelial cells co-expressing nestin and Ki-67. Microvessel proliferation was estimated by vascular proliferation index (VPI), the ratio of vessels containing immature proliferating endothelial cells, and the total number of immature vessels. VPI was evaluated in 178 breast cancer tissue sections. High VPI showed significant association to several markers of aggressive breast cancer, including negative estrogen receptor (ER) status (p=0.003), high tumour cell proliferation by Ki-67 (p=0.004), high p53 expression (p=0.001), five immunohistochemical profiles for the basal-like phenotype (odds ratios (OR); range 2.8-6.3), and the triple negative phenotype (TNP)(p=0.040). Concerning the mode of detection, high VPI was three times more likely to be presented in an interval detected breast cancer compared with a screening detected (OR: 3.0, p<0.0005). Both in univariate and multivariate analysis, high VPI was significantly associated with poor survival (p=0.034 and p=0.022, respectively). In conclusion, activated angiogenesis, estimated by microvessel proliferation, is associated with several markers of aggressive breast cancer phenotype, basal-like breast cancer, interval detection, and a significant predictor of prognosis in this series of breast cancer. 1. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell. 1990 Feb 23;60(4):585-95. 2. Mokry J, Cizkova D, Filip S, Ehrmann J, Osterreicher J, Kolar Z, et al. Nestin expression by newly formed human blood vessels. Stem Cells Dev. 2004 Dec;13(6):658-64. 3. Mokry J, Ehrmann J, Karbanova J, Cizkova D, Soukup T, Suchanek J, et al. Expression of intermediate filament nestin in blood vessels of neural and non-neural tissues. Acta Medica (Hradec Kralove). 2008;51(3):173-9. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1372. doi:1538-7445.AM2012-1372

Hematopoietic, CNS And Skeletal Muscle Stem Cells As Drug Targets: Opportunities, Progress And Challenges

Lineage-committed stem and progenitor cells are currently targeted by a handful of medicines, mainly to treat conditions involving the immune and hematopoietic systems. Knowledge of new stem and progenitor cell populations in the body is accumulating at a rapid pace and a new era of targeting resident stem cell populations for therapeutic ends is coming into focus. Small-molecule regulators of body-resident stem and progenitor cell assess are now a reality both in the clinic and as promising new drugs in the development pipeline. This review will explore the current state of the art with an emphasis on emerging concepts and experimental systems in the therapeutic regulation of endogenous stem and progenitor cell populations.