Sharareh Sharififar

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes. This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

Establishing embryonic mouse neural stem cell culture using the neurosphere assay.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum-free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies < 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are < 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated.

The effects of repetitive transcranial magnetic stimulation on proliferation and differentiation of neural stem cells

Repetitive transcranial magnetic stimulation (rTMS) is a new method for treating many neurological conditions; however, the exact therapeutic mechanisms behind rTMS-induced plasticity are still unknown. Neural stem and progenitor cells (NS/PCs) are active players in brain regeneration and plasticity but their behavior in the context of rTMS therapy needs further elucidation. We aimed to evaluate the effects of rTMS on proliferation and differentiation of NS/PCs in the subventricular zone (SVZ) of adult mouse brain. Adult male mice (n=30) were divided into rTMS (1-Hz and 30-Hz) and sham groups and treated for 7 or 14 consecutive days. Harvested NS/PCs from the SVZ were cultured in the neurosphere assay for 8 days and the number and size of the resulting neurospheres as well as their in vitro differentiation capacity were evaluated. After one week of rTMS treatment at 1-Hz and 30-Hz compared with sham stimulation, the mean neurosphere forming frequency per brain was not different while this measure significantly increased after two weeks (P<0.05). The mean neurosphere diameter in 1-Hz treatment paradigm was significantly larger compared with sham stimulation at both 1 and 2 weeks. In contrast, 30-Hz treatment paradigm resulted in significantly larger neurospheres only after 2 weeks. Importantly, rTMS treatment at both frequencies increased neuronal differentiation of the harvested NS/PCs. Furthermore, one week in vitro rTMS treatment of NS/PCs with both 1-Hz and 30-Hz increased NS/PCs proliferation and neuronal differentiation. It is concluded that both 1-Hz and 30-Hz rTMS treatment increase NS/PCs proliferation and neuronal differentiation.

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

Purifying Immature Neurons from Differentiating Neural Stem Cell Progeny Using a Simple Shaking Method

Objective: Enriched neuronal cell populations are valuable tools both for laboratory investigations and cell therapy applications. However, available cell purifying approaches demand costly equipment such as FACS or MACS that limits their universal accessibility. In this study, we developed an efficient method for purifying immature neuronal cells from differentiating neural stem cell (dNSC) progeny based on their differential substrate attachment properties without using any expensive cell separating tools. Methods: Neural stem cells were harvested from the ganglionic eminence of embryonic day 14 mice brains using the neurosphere assay. Neurospheres were then dissociated into single cells and differentiated employing the neuroblast assay method. Following a brief trypsinization, the dNSC culture was gently shaken at 150 rpm for 30 min to detach the top neuronal cell clusters from the underlying astrocytic cell monolayer. The neuronal purification yield, astrocyte contamination, and presence of dividing cells were compared to a MACS purification method using PSANCAM antibody. Results: While a neuronal yield of 97.1 ± 0.45% was achieved using MACS; it reached 97.9 ± 0.6% using the shaking method that was not significantly different.On the other hand, the percentage of astrocytes in the MACS approach was 1.18 ± 0.15%, but it significantly decreased to 0.6 ± 0.15% using the shaking method. Moreover, 4.41 ± 0.23% and 5.3 ± 0.4% of the isolated cells in the MACS and shaking methods, respectively, were Ki-67 immunoreactive dividing cells, of which 97.34 ± 1.6% and 97.9 ± 0.7% were co-expressing β III-tubulin, confirming their neuronal identity. Additionally, based on the neural-colony forming cell assay, the shaking method resulted in the generation of a homogenous neuronal cell population without any bona fide NSC contamination. Conclusions: The shaking purification method allows easy, low cost, efficient and large-scale separation of immature neurons from dNSC progeny, potentially benefiting both basic and clinical applications.

Depletion of neural stem cells from the subventricular zone of adult mouse brain using cytosine b‐Arabinofuranoside

Neural stem cells (NSCs) reside along the ventricular axis of the mammalian brain. They divide infrequently to maintain themselves and the down‐stream progenitors. Due to the quiescent property of NSCs, attempts to deplete these cells using antimitotic agents such as cytosine b‐Aarabinofuranoside (Ara‐C) have not been successful. We hypothesized that implementing infusion gaps in Ara‐C kill paradigms would recruit the quiescent NSCs and subsequently eliminate them from their niches in the subventricular zone (SVZ).

Poster 10: Trajectories of PROMIS Function and Well-Being Measures in Morbidly Obese Patients After Orthopedic Trauma

Disclosures: Conan So: I Have No Relevant Financial Relationships To Disclose Objective: Traditionally, illness severity, social factors, and comorbid conditions have been examined as predictors of hospital outcomes. However, recent research in the rehabilitation setting demonstrated that physical function outperformed co-morbidity indices as a predictor of 30-day readmission. The purpose of this study was to review the literature examining the association between acute hospital physical function and various hospital outcomes. Design: A literature review of the MEDLINE database using PubMed was performed. Search terms included acute functional outcomes and frailty outcomes. Studies were included if they were in English and examined how functional metrics collected at acute care hospitalization impacted clinical outcomes. Cohort characteristics and measures of associations were extracted from the studies. Setting: Acute Care Hospital Participants: Not applicable Interventions: Not applicable Main Outcome Measures: Hospital readmission, length of stay, mortality, discharge location, and physical function post-acute care Results: 31 eligible studies were identified (hospital readmissions: 6; discharge locations: 11; length of stay: 4; mortality: 15; function: 6). 13 different metrics were used to assess function. Higher function during acute care was associated with statistically significant lower odds of hospital readmission, discharge to skilled nursing facilities and inpatient rehabilitation facilities, shorter hospital stays, decreased mortality, and greater functional recovery when compared to patients with lower function, when adjusted for age and sex. 88% (23/26) of studies that reported measures of association exhibited a statistically significant relationship between function and hospital outcomes. Conclusions: Although this review was limited by the heterogeneity of functional measures and hospital outcomes, there is a growing body of evidence that functional metrics assessed in acute care are significant predictors of a broad array of hospital outcomes. Future research would benefit from more widespread collection of functional data and consensus of functional metrics for use in acute care hospitals. Level of Evidence: Level V

Pre-injury depression and anxiety in patients with orthopedic trauma and their treatment

PURPOSE Depressive symptoms have a known negative impact on outcomes following musculoskeletal injury. This study determined the pre-injury prevalence of psychiatric diagnoses of depression and anxiety, medication lapses and psychiatric consult services among patients admitted for orthopaedic trauma. METHODS This is a retrospective study of data from our Level-1 trauma center. Patients admitted to the orthopaedic trauma service during 2010-2015 were included (N = 4053). Demographics, Injury Severity Scores (ISS), mental health diagnoses, psychotropic medications, medication type and delay, psychiatric consultation use, intensive care unit (ICU) stay and total hospital length of stay (LOS) were abstracted from medical records and the institutional trauma registry. RESULTS The 12-month prevalence of a major depressive episode is 6.6%-8.6% in adults in the United States. In our database, only 152/4053 (3.8%) of the patients had documented medical history of depression (80%) or anxiety (30%), and these patients had a 32% longer LOS (p < 0.016). Nearly two-thirds of patients who used psychotropic medications prior to injury experienced a delay in receiving these medications in the hospital (median = 1.0 day, range 0-14 days). Sixteen percent of patients also received a new psychotropic medication while hospitalized: an antipsychotic (8/16 patients, to treat delirium), an anxiolytic (3/16 patients for acute anxiety), or an antidepressant (1/16). Among patients with depression or anxiety, 16.7% received a psychiatric consult. Patients with psychiatric consults had higher ISS, were more likely to have longer ICU LOS and had longer hospital LOS than those without consults (all p < 0.05). CONCLUSION The prevalence of depression and anxiety is grossly under-reported in our registry compared to national prevalence data. Patients with pre-existing disease had longer LOS and a higher rate of extended ICU care. Further studies are needed to characterize the true prevalence of disease in this patient population and its effect on patient outcomes after traumatic orthopaedic injury.

Adding electrical stimulation during standard rehabilitation after stroke to improve motor function. A systematic review and meta-analysis

BACKGROUND Clinical studies have shown that sensory input improves motor function when added to active training after neurological injuries in the spinal cord. OBJECTIVE We aimed to determine the effect on motor function of extremities of adding an electrical sensory modality without motor recruitment before or with routine rehabilitation for hemiparesis after stroke by a comprehensive systematic review and meta-analysis. METHODS We searched databases including MEDLINE via PubMed and the Cochrane Central Register of Controlled Trials from 1978 to the end of November 2017 for reports of randomized controlled trials or controlled studies of patients with a clinical diagnosis of stroke who underwent 1) transcutaneous electrical nerve stimulation (TENS) or peripheral electromyography-triggered sensory stimulation over a peripheral nerve and associated muscles or 2) acupuncture to areas that produced sensory effects, without motor recruitment, along with routine rehabilitation. Outcome measures were motor impairment, activity, and participation outcomes defined by the International Classification of Functioning, Disability and Health. RESULTS The search yielded 11studies with data that could be included in a meta-analysis. Electrical sensory inputs, when paired with routine therapy, improved peak torque dorsiflexion (mean difference [MD] 2.44 Nm, 95% confidence interval [CI] 0.26-4.63). On subgroup analysis, the combined therapy yielded a significant difference in terms of sensory stimulation without motor recruitment only on the Timed Up and Go test in the chronic phase of stroke (MD 3.51sec, 95% CI 3.05-3.98). The spasticity score was reduced but not significantly (MD-0.83 points, 95% CI -1.77-0.10). CONCLUSION Electrical sensory input can contribute to routine rehabilitation to improve early post-stroke lower-extremity impairment and late motor function, with no change in spasticity. Prolonged periods of sensory stimulation such as TENS combined with activity can have beneficial effects on impairment and function after stroke.