Human Neuronal Stem Cells Research Articles

Neuronal specific Caveolin‐1 overexpression enhances dendritic growth and arborization in differentiated human neuronal stem cells derived from induced pluripotent stem cells

IntroductionNeuroplasticity is evoked by extracellular signals that bind receptors in the plasma membrane to induce signal transduction that results in polarized actions in the neuronal plasma membrane. Membrane/lipid rafts (MLR) and cholesterol binding protein caveolin‐1 (Cav‐1) provide a signaling membrane platform that tethers and regulates cytoskeletal components necessary for proper dendritic growth, maintenance and neuroplasticity. Our lab has demonstrated in rodents primary neurons that overexpression of neuronal cav‐1 enhances pro‐growth/pro‐survival signaling, and improves dendritic growth and arborization. We utilized neurons derived from human induced pluripotent stem cells (iPSCs) to ask whether Cav‐1 controls similar signaling and functions in human neurons as it does in rodent neurons. We overexpressed Cav‐1 driven by the neuronal specific synapsin promoter (SynCav1) and evaluated changes in dendritic morphology after the differentiation process.MethodsiPSC derived neuronal stem cells (NSCs) were cultured in DMEM/F12+Gluamax media with N‐2 supplement, B‐27 supplement, Pen/Strep, and basic Fibroblast Growth Factor (bFGF). Neuronal differentiation was started by bFGF withdrawal from media. NSCs were transfected with either SynCav1 or SynGFP as control when differentiation was started. Cells were fixed 3 weeks after differentiation and stained for Cav‐1, the dendritic marker microtubule‐associated protein 2 (MAP2), and the axonal marker neurofilament (SMI‐31). Dendritic length, branching (arborization), area, and volume were measured by Autoneuron (MBF Bioscience), a tracing algorithm that measures 3D image volume stacks. Cells were also collected and homogenized to measure Cav‐1 levels via western blots.ResultsWestern blots showed that Cav‐1 was increased in the cells transfected with SynCav‐1 compared to SynGFP 3 weeks after differentiation. Compared with SynGFP, SynCav1 transfection significantly enhanced dendritic length (p=0.002), branching points/arborization (p=0.0004), dendrite area (p=0.003), and dendrite volume (p=0.009)ConclusionNeuron‐targeted expression of Cav‐1 significantly enhanced dendritic growth of human iPSC‐derived neurons suggesting the presence of efficient Cav‐1 dependent signaling in these cells.

A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC).

We present here a microfluidic device that generates sub-millimetric hollow hydrogel spheres, encapsulating cells and coated internally with a layer of reconstituted extracellular matrix (ECM) of a few microns thick. The spherical capsules, composed of alginate hydrogel, originate from the spontaneous instability of a multi-layered jet formed by co-extrusion using a coaxial flow device. We provide a simple design to manufacture this device using a DLP (digital light processing) 3D printer. Then, we demonstrate how the inner wall of the capsules can be decorated with a continuous ECM layer that is anchored to the alginate gel and mimics the basal membrane of a cellular niche. Finally, we used this approach to encapsulate human Neural Stem Cells (hNSC) derived from human Induced Pluripotent Stem Cells (hIPSC), which were further differentiated into neurons within the capsules with negligible loss of viability. Altogether, we show that these capsules may serve as cell micro-containers compatible with complex cell culture conditions and applications. These developments widen the field of research and biomedical applications of the cell encapsulation technology.

Reparative effects of neural stem cells in neonatal rats with hypoxic–ischemic injury are not influenced by host sex

BACKGROUNDGender is increasingly recognized as an important influence on brain development, disease susceptibility, and response to pharmacologic/rehabilitative treatments. In regenerative medicine, it remains entirely unknown whether there is an interaction between transplanted stem cells and host gender that might bias efficacy and safety in some patients but not others.METHODSWe examined the role of recipient gender in a neonatal rat hypoxia-ischemic injury (HII) model, treated with human female neural stem cells (hNSCs), labeled with superparamagnetic iron-oxide (SPIO) particles implanted into the contralateral cerebral ventricle. We monitored HII evolution (by MRI, histopathology, behavioral testing) and hNSC fate (migration, replication, viability).RESULTSRecipient gender after implantation did not influence the volume or location of ischemic injury (1, 30, or 90d) or behavior (90d). SPIO labeling did not influence HII evolution. Implantation had its greatest benefit on mild/moderate injuries which remained stable rather than increasing as in severe HII as is the natural history for such lesions.CONCLUSIONSOur results suggest that hNSC treatment (including using hNSCs that are pre-labeled with iron to allow tracking in real time by MRI) would be equally safe and effective for male and female human newborns with mild-to-moderate HII.

EPA-0324 – Role of Glucocorticoid receptors in Mood Disorders

This tall will review the work conducted within Moodinflame around the role of the glucocorticoid receptor in both cellular models of neurogenesis and in clinical samples of depressed patients. In our established model of ‘depression in a dish’, using human neuronal stem cells, we have found that the glucocorticoid receptor is the target of both glucocorticoid (stress) hormones and of antidepressants, with the ability of both inhibit and stimulate neurogenesis in the hippocampus. In addition, reduced glucocorticoid receptor function and expression in the peripheral blood depressed patients is a consistent finding across clinical samples as diverse as young adults, older depressed patients with coronary heart diseases, and patients with cytokine-induced depression. This body of research confirms the pivotal role of the glucocorticoid receptor, and the potential clinical relevance of targeting it for therapeutic antidepressant purposes.

Investigation of the Competition Between Cell/Surface and Cell/Cell Interactions During Neuronal Cell Culture on a Micro‐Engineered Surface

To investigate the respective roles of topography and cell/cell interactions in the development of a guided neuronal network on an engineered surface, micropatterned PDMS substrates were generated with different microgrooves geometry and investigated for the influence of cell density on neurite outgrowth and alignment. Through this systematic investigation, using a human neuronal stem cell line, the rules of neuronal network development and guiding could be learned. The results show that when cell density is increased the influence on neuritic outgrowth and alignment is very different for the various grooves geometries. The data emphasized the competition, in neurite development, between physical cues brought by surface topographical features and cell to cell communications. These results can be of particular interest for designing functional neuronal networks with a controlled architecture.

Neural Stem Cell-Mediated Enzyme/Prodrug Therapy for Glioma

Glioblastoma (GBM), the most common primary brain tumor in adults, portends a grim prognosis with median survival rates typically less than a year.1 The aggressive, infiltrative nature of GBM precludes a surgical cure, and the blood-brain barrier has limited the efficacy of systemic chemotherapeutic interventions. Previous research has demonstrated the tumor-tropic behavior of neuronal stem cells (NSC), whereby these cells preferentially aggregate to glioma foci when implanted in the brain.2 Thus, stem cells have become an attractive tumor-specific delivery device surmounting the limitations of surgery or chemotherapyin the treatment of high grade glioma.3 Recent research published in this month’s issue of Science Translational Medicine by Aboodyet al, has exploited the tumor localizing characteristics of NSC and tested a novel enzyme/prodrug approach for the treatment of gliomas.4 Utilizing a cytosine deaminase (CD) expressing-human NSC line, HB1.F3.CD, the group hypothesized that the stem cells would co-localize to tumor cells and convert a systemically administered prodrug, 5-fluorocytosine (5-FC) to the active, nucleic acid analog 5-fluorouracil (5-FU), thereby leading to cell death. Their results demonstrate a potential breakthrough in the treatment of this most challenging pathology. To begin, the team created a stable human NSC line from HB1.F3 using a transgene for CD, then expanded this colony for all future experiments. They subsequently assessed the immunogenicity of the HB1.F3.CD NSC line and determined that they expressed HLA class I antigens but not class II antigens. Therefore, their cell line was unlikely to induce an immunogenic response, a caveat that has plagued other attempts in utilizing this approach. The investigators then evaluated the tropism of their NSC in a murine model under different simulated conditions that human patients experience. Specifically, they determined that when implanted into mouse brains, their NSC co-localized to glioma cells, despite the mouse brains having been previously irradiated or treated with corticosteroids. Additionally, their NSC were able to migrate to the tumor sites when injected adjacent to the tumor or in the opposite hemisphere, regardless of prior radiation or corticosteroid therapy. The safety profile and the tumorgenicity of their NSC line was then evaluated. Using a murine model injected intracranially with HB1.F3.CD, they wanted to determine if their NSC’s were independently tumorogenic and whether there was a maximum tolerated dose of NSC HB1.F3.CD. At both 4 and 12 weeks post NSC implantation, the mice were euthanized and their organs harvested. All organ systems failed to demonstrate any evidence of human NSC, and no tumors were discovered regardless of the amount of NSC injected. Having demonstrated tumor tropism and safety, the team analyzed the in vivo efficacy of their co-localizing NSC to preferentially kill the surrounding, dividing tumor cells. Mice were injected with glioma cells followed by the HB1.F3.CD NSC’s 7 days later. Peritoneal injections of 5-FC were performed on days 11 through 17, and the brains were evaluated. Typical response to treatment demonstrated extensive areas of tumor necrosis surrounded by areas of residual viable tumor (Figure). The therapeutic efficacy was then determined by measuring the viable brain tumor after 1 round of treatment with 5-FC in 24 mice. Their volumetric analysis demonstrated that mice receiving the NSC/5-FC combination therapy had a significantly smaller tumor burden than those mice that did not receive combination therapy.Figure: Diagramof CD-expressing NSCs localized to tumor cells, and CD conversion of 5-FC to 5-FU, which readily diffuses out of the NSCs to selectively kill surrounding, dividing tumor cells. (B and C) H&E-stained brain tumor sections from U251 glioma-bearing mice that received HB1.F3.CD NSCs only (B) or HB1.F3.CD NSCs in combinationwith 5-FC (C). White arrows indicate tumor region.NSCs-neural stem cells. CD-cytosine deaminase. Modified from Aboody KS, Najbauer J, Metz MZ, et al. Neural stem cell -mediated enzyme/prodrug therapy for glioma: preclinical studies. Sci Transl Med. 2013;5(184):184ra59. Reprinted with permission from AAAS.In conclusion, the researchers provide convincing evidence that an NSC-prodrug delivery system to combat glioblastoma is safe, effective and feasible in a murine model. These investigators have provided the necessary pilot data to translate these experimental results from the benchtop to the bedside in the first human clinical trial (NCT01172964) using a NSC-prodrug delivery system in patients with recurrent high-grade glioma. Future research in this area will hopefully offer new therapeutic options to treat this challenging disease.

Combined Transplantation of Human Neuronal and Mesenchymal Stem Cells following Spinal Cord Injury

Transplantation of human fetal neural stem cells (hNSCs) previously demonstrated significant functional recovery after spinal cord contusion in rats. Other studies indicated that human mesenchymal stem cells (hMSCs) can home to areas of damage and cross the blood–brain barrier. The purpose of this article is to determine if combined administration of mesenchymal stem cells and neuronal stem cells improves functional outcomes in rats. The study design was a randomized controlled animal trial. Female adult Long-Evans hooded rats underwent laminectomy at T10 level. Moderate spinal cord contusion at T10 level was induced by the MASCIS Impactor. Four groups were identified. The MSC + NSC group received hMSCs intravenously (IV) immediately after spinal cord injury (acute) and returned 1 week later (subacute) for injection of hNSC directly at site of injury. The MSC-only group received hMSC IV acutely and cell media subacutely. The NSC-only group received cell media IV acutely and hNSC subacutely. The control group received cell media IV acutely and subacutely. Subjects were assessed for 6 weeks using Basso, Beattie, Bresnahan Locomotor Rating Score. Twenty-four subjects were utilized, six subjects in each group. Statistically significant functional improvement was seen in the MSC + NSC group and the NSC-only group versus controls (p = 0.027, 0.042, respectively). The MSC-only group did not demonstrate a significant improvement over control (p = 0.145). Comparing the MSC + NSC group and the NSC-only group, there was no significant difference (p = 0.357). Subacute transplantation of hNSCs into contused spinal cord of rats led to significant functional recovery when injected either with or without acute IV administration of hMSCs. Neither hMSCs nor addition of hMSC to hNSC resulted in significant improvement.

Quantifying the protective effect of antioxidants on metabolic function in human neuronal stem cells

Quantifying the protective effect of antioxidants on metabolic function in human neuronal stem cells

Neuroprotection by human umbilical cord blood-derived progenitors in ischemic brain injuries.

Stem cells have an extremely high potential to treat many devastating diseases, including neuronal injuries. Albeit the need for human neuronal stem cells, their quantities are very limited by relying on early human embryos as the main source. Therefore, progenitors of other origins, such as human umbilical cord blood (CB) are being considered. In the last decade, various populations isolated from the CB were reported to differentiate in vitro towards a neural phenotype. The conditions to induce the cell differentiation are not conclusive and may include addition of chemicals, cytokines and growth factors, including the nerve growth factor (NGF). Some CB cells were found to express the TrkANGF receptor, suggesting an endogenous role for this growth factor also in the CB environment. The ability of CB and derived stem cell populations to protect against neurological deficits was shown, both in vitro and in vivo, in models of ischemic brain injuries. In rodent models of stroke, heatstroke, brain trauma and brain damage at birth, CB cells either by intravenous injection or intrastriatal transplantation, were found to reduce the infarct size and the neurological deficits caused by the injury. The restorative effects of CB were suggested to be mediated by mechanisms other than cell replacement. Some of the proposed mechanisms involve reduced inflammation, nerve fiber reorganization by trophic actions, increased cell survival and enhanced angiogenesis. Furthermore, treatment with CB was found to have a therapeutic window of days compared with the present 36 hour window for the treatment of stroke with clinically available tools such as recombinant tissue plasminogen activator. Considering the encouraging results with whole CB and derived cells transplantation in ischemic injury models and since CB is widely available and have been used clinically, they may be an excellent source of cells for treatment of human brain ischemic disorders.

Markers Distinguishing Cancer Stem Cells from Normal Human Neuronal Stem Cell Populations in Malignant Glioma Patients

Markers Distinguishing Cancer Stem Cells from Normal Human Neuronal Stem Cell Populations in Malignant Glioma Patients

Optimization and Validation of FePro Cell Labeling Method

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12–16 hours) incubation time and uses relatively high dose of Pro (5–6 µg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 µg/ml and Pro 0.75 to 3 µg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 µg/ml of Fe and 3 µg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI.

A simple and efficient method for generating Nurr1-positive neuronal stem cells from human wisdom teeth (tNSC) and the potential of tNSC for stroke therapy

We have isolated human neuronal stem cells from exfoliated third molars (wisdom teeth) using a simple and efficient method. The cultured neuronal stem cells (designated tNSC) expressed embryonic and adult stem cell markers, markers for chemotatic factor and its corresponding ligand, as well as neuron proteins. The tNSC expressed genes of Nurr1, NF-M and nestin. They were used to treat middle cerebral artery occlusion (MCAO) surgery-inflicted Sprague-Dawley (SD) rats to assess their therapeutic potential for stroke therapy. For each tNSC cell line, a normal human impacted wisdom tooth was collected from a donor with consent. The tooth was cleaned thoroughly with normal saline. The molar was vigorously shaken or vortexed for 30 min in a 50-mL conical tube with 15-20mL normal saline. The mixture of dental pulp was collected by centrifugation and cultured in a 25-cm(2) tissue culture flask with 4-5mL Medium 199 supplemented with 5-10% fetal calf serum. The tNSC harvested from tissue culture, at a concentration of 1-2x10(5), were suspended in 3 microL saline solution and injected into the right dorsolateral striatum of experimental animals inflicted with MCAO. Behavioral measurements of the tNSC-treated SD rats showed a significant recovery from neurologic dysfunction after MCAO treatment. In contrast, a sham group of SD rats failed to recover from the surgery. Immunohistochemistry analysis of brain sections of the tNSC-treated SD rats showed survival of the transplanted cells. These results suggest that adult neuronal stem cells may be procured from third molars, and tNSC thus cultivated have potential for treatment of stroke-inflicted rats.

Mass spectrometry based proteomic analysis of human stem cells: a brief review

Stem cells can give rise to various cell types and are capable of regenerating themselves over multiple cell divisions. Pluripotency and self-renewal potential of stem cells have drawn vast interest from different disciplines, with studies on the molecular properties of stem cells being one example. Current investigations on the molecular basis of stem cells pluripotency and self-renewal entail traditional techniques from chemistry and molecular biology. In this mini review, we discuss progress in stem cell research that employs proteomics approaches. Specifically, we focus on studies on human stem cells from proteomics perspective. To our best knowledge, only the following types of human stem cells have been examined via proteomics analysis: human neuronal stem cells, human mesenchymal stem cells, and human embryonic stem cells. Protein expression serves as biomarkers of stem cells and identification and expression level of such biomarkers are usually determined using two-dimensional electrophoresis coupled mass spectrometry or non-gel based mass spectrometry.

2‐DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM)

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.

Down-regulation of MHC class I expression in human neuronal stem cells using viral stealth mechanism

Due to their unique capacity for self-renewal in addition to their ability to differentiate into cells of all neuronal lineages, neuronal stem cells (NSCs) are promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. However, there are few studies on immune rejection, which is one of the main problems facing successful stem cell therapy. In order to determine if human NSC might be rejected after transplantation the MHC expression level was examined in the HB1.F3 cell line, which has previously been shown to exhibit NSC properties. The results showed low expression levels of the MHC class I molecules on the surfaces of these cells. A dramatic increase in the MHC class I expression level was observed when the cells were treated with IFN-γ, TNF-α, and IL-1β, alone or in combination. The maximum induction of MHC class I protein expression was observed at above 20 ng/ml IFN-γ 48 h after the treatment. The apparent additive effects of TNF-α and IL-1β in combination on the maximum induction of MHC class I expression exerted by IFN-γ treatment were not observed. The MHC class I levels elevated by IFN-γ were sustained for 72 h after withdrawing the IFN-γ. Therefore, this study introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce the MHC class I expression level on the cell surface after infection, into HB1.F3 cells. The cells transfected with the hCMV US2, US3, US6 or US11 genes showed 20–50% reduction in the MHC class I expression level compared with the mock-transfected cells. These results suggest that NSC expresses high levels of the MHC class I proteins, and unless they are modified, might be rejected upon transplantation. In addition, the various viral stealth mechanisms can be exploited for stem cell transplantation.

홍삼추출액의 인간성체신경줄기세포 증식과 세포사 관련 세포주기의 변화에 대한 효과

홍삼추출액의 신경성체줄기세포 성장과 생존에 미치는 영향을 분석하고자 human neuronal stem cell line인 F3 cell을 배양한 후 홍삼추출액을 여러 농도로 희석하여 MTT assay로 cell viability를 측정하였고 FACS analysis로 cell Cycle변화를 측정하였다. 특정한 농도에서는 세포가 증식되는 경향을 보였으며 농도가 증가되면서 viability가 감소되는 현상을 확인할 수 있었다. Cell cycle 분석상 세포증식시에는 5 phase 및 G2/M phase가 증가되는 경향을 보였고, viability가 감소되면서 S phase가 감소되고 G0/G1 phase 가 증가 되었다. 한편 DNA fragmentation이 cell viability감소에 따라 증가되었으나, Caspase 3 activation 또는 Bax expression과는 관련성이 적었다. The present study is to determine whether the Red-ginseng extract has a proliferative or cytotoxic effect on the human neuronal stem cells(hNSCs). The hNSCs were grown and incubated with different doses of Red-ginseng extract. We tested the proliferative or cytotoxic effects by MTT and FACS analysis. Cell viability cell cycle analysis, DNA fragmentation, and bax or PARP expressions were evaluated. The hNSCs showed a proliferafe trend with its peak concentration at 0.3 <TEX>$\mu\textrm{g}$</TEX>/<TEX>$m\ell$</TEX>. Beyond this point, higher doses decreased viabilities and showed a cytotoxic effect at 10 <TEX>$\mu\textrm{g}$</TEX>/<TEX>$m\ell$</TEX>. There was a tendency of increased S and G2/M phases during cell proliferation. In a cytotoxic condition, decreased S phase and increased G0/G1 phases were noted, suggesting cell cycle arrest. The cytotoxic effect was associated with increase DNA fragmentation in a dose-dependent manner, However PARP cleavage or bax expression was not detected. Our results suggest that Red-ginseng extract has dual effects, the cell proliferative or cytotoxic effect, on hNSCs in vitro with dose-dependent manner.

Isolation and characterization of normal adult human epithelial pluripotent stem cells.

Both reproductive and therapeutic cloning of human stem cells have been made possible with recent technological advances in the isolation of embryonic stem cells and of pluripotent stem cells from adult tissues. We have isolated normal human kidney and human breast epithelial stem cells, as well as having characterized "immortalized" cells from human neuronal and human pancreatic tissue (Trosko et al., Methods 20:245-264, 2000). The isolation was motivated by the stem cell theory of carcinogenesis. Based on the assumption that stem cells would not express connexin genes, nor have functional gap junctional intercellular communication (GJIC), we have demonstrated that the human kidney, breast, neuronal, and pancreatic stem cells can divide either symmetrically or asymmetrically, depending on whether they are grown in microenvironmental conditions that suppress GJIC (the undifferentiated, proliferative state) or induce GJIC (the differentiated state). Normal breast epithelial stem cells appear to be intrinsically "immortal" until induced to express GJIC, at which time, with appropriate substrate and microenvironmental nutrients, they can form three-dimensional "organoids." expressing markers associated with the mature mammary tissue and forming a physical structure very similar to the in vivo budding, ductal structures. The breast stem cells can be prevented from "mortalizing" and can be converted to neoplastic cells, which maintain many phenotypes of the stem cells.

Waisman Center

The Waisman Center MRDDRC supports behavioral, social, and biomedical research on human development, developmental disabilities, and neurodegenerative diseases. Examples of research conducted in each of the four MRDDRC units include: abnormalities in gene expression in human neuronal stem cells and progenitor cells from Down syndrome tissue (from the Molecular and Genetic Sciences Unit), language disorders in adolescents with Down syndrome and Fragile X syndrome (from the Communication Processes Unit); the development of selective visual attention during the first 6 months of life (from the Sensory and Cognitive Processes Unit); and the long-range effects of early intervention on special education placement, delinquency, and high school graduation rates (from the Social and Affective Processes Unit).

Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study

Identification of genes and characterisation of their function is an essential step towards understanding complex pathophysiological abnormalities in Down's syndrome. We did a study to investigate abnormalities in gene expression in human neuronal stem cells and progenitor cells from Down's syndrome and control post-mortem human fetal tissue. Indexing-based differential display PCR was done on neuronal precursor cells derived from the cortex of a fetus with Down's syndrome, and findings were compared with those of two control samples. Findings were validated against neurosphere preparations from three independent Down's syndrome fetuses and five independent controls by real-time quantitative PCR. Results of differential display PCR analysis showed that SCG10--a neuron--specific growth-associated protein regulated by the neuron-restrictive silencer factor REST-was almost undetectable in the Down's syndrome sample. This finding was validated by real-time PCR. We also found that other genes regulated by the REST transcription factor were selectively repressed, whereas non-REST-regulated genes with similar functions were unaffected. Changes in expression of several key developmental genes in the Down's syndrome stem-cell and progenitor-cell pool correlated with striking changes in neuron morphology after differentiation. Our findings suggest a link between dysregulation of the REST transcription factor and some of the neurological deficits seen in Down's syndrome. Experimental REST downregulation has been shown to trigger apoptosis, which could account for the striking and selective loss of neurons in the differentiated Down's syndrome cell preparations.

In vivo hematopoietic potential of human neuronal stem cells

Transplantation in primary and secondary fetal sheep recipients was used to access the in vivo hematopoietic potential of human neuronal stem cells (NSC). Eleven primary recipients were transplanted with NSC (8×105 cells/fetus) and analyzed for hematopoietic chimerism at 60 days and at 6, 8 and 15 months post-transplant. At 60 days post-transplant human cells were detected in liver, muscle, spleen, thymus and bone marrow. However, at all later time points flow cytometric analysis of PB and BM revealed the presence of human multilineage hematopoietic engraftment but not CD34+ cells. PCR analysis confirmed the presence of human cells in PB and BM and further demonstrated the presence of human cells in liver, thymus, and spleen. At 6 months post-transplant primary sheep were mobilized with G-CSF (5mcg/Kg). The mobilization pattern of sheep engrafted with NSC different from those engrafted with human BM HSC. Unlike HSC sheep, in NSC animals, except for the appearance of 0.12% CD34+ cells in PB on day 3, human cell activity remained low in both BM and PB throughout mobilization. Clonogenic assay of BM cells at 8 months revealed that of the 53 hematopoietic colonies analyzed on day 19 of culture, 8 colonies were of human origin by both karyotype and PCR. At 15 months post-transplant human CD45+/HLA-DR+ cells were obtained from BM of primary sheep by sorting and transplanted into secondary recipients. FACS and PCR analyses of secondary recipients demonstrated the presence of human cells inthe BM and Spleen. These results demonstrate the hematopoietic potential of human NSC and suggest that their hematopoietic activity may be regulated differently from those of human HSC in this xenogeneic model of human hematopoiesis.