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Simplified 3D protocol capable of generating early cortical neuroepithelium.

ABSTRACT: Here, we report a 3D cerebellar differentiation protocol with quick startup method, defined medium and no special materials or handling requirements. Three fibroblast growth factors (FGF2, 4 and 8) were used for cerebellar patterning and smoothened agonist (SAG) for granule cell development. After 35?days, differentiation products exhibited similar structures and neuronal markers reported in prior 'organoid' and 'spheroid' protocols. This included cells positive for KIRREL2 (a marker of early cerebellar neuroepithelium) and ZIC1 (a marker for granule cells). Follow-up tests indicated that addition of FGFs, if helpful, was not required to generate observed structures and cell types. This suggests that intrinsic production of patterning factors by aggregates themselves may be adequate for region-specific 3D modeling. This protocol may be used as a quick, easy and cost-efficient method for 3D culture, whether to research development of the early cerebellar neuroepithelium, a base to generate mature cortical structures, or to optimize minimal-factor protocols for other brain regions.

SUBMITTER: Holmes DB

PROVIDER: S-EPMC5374398 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Simplified 3D protocol capable of generating early cortical neuroepithelium.

Holmes Dwayne B DB Heine Vivi M VM

Biology open 20170315 3

Here, we report a 3D cerebellar differentiation protocol with quick startup method, defined medium and no special materials or handling requirements. Three fibroblast growth factors (FGF2, 4 and 8) were used for cerebellar patterning and smoothened agonist (SAG) for granule cell development. After 35 days, differentiation products exhibited similar structures and neuronal markers reported in prior 'organoid' and 'spheroid' protocols. This included cells positive for KIRREL2 (a marker of early ce ...[more]

PMID: 28167491

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Project description:OBJECTIVE:To assess the efficacy of a simple, goal-directed sepsis treatment protocol for reducing mortality in patients with severe sepsis in Zambia. DESIGN:Single-center nonblinded randomized controlled trial. SETTING:Emergency department, ICU, and medical wards of the national referral hospital in Lusaka, Zambia. PATIENTS:One hundred twelve patients enrolled within 24 hours of admission with severe sepsis, defined as systemic inflammatory response syndrome with suspected infection and organ dysfunction INTERVENTIONS:: Simplified Severe Sepsis Protocol consisting of up to 4 L of IV fluids within 6 hours, guided by jugular venous pressure assessment, and dopamine and/or blood transfusion in selected patients. Control group was managed as usual care. Blood cultures were collected and early antibiotics administered for both arms. MEASUREMENTS AND MAIN RESULTS:Primary outcome was in-hospital all-cause mortality. One hundred nine patients were included in the final analysis and 88 patients (80.7%) were HIV positive. Pulmonary infections were the most common source of sepsis. In-hospital mortality rate was 64.2% in the intervention group and 60.7% in the control group (relative risk, 1.05; 95% CI, 0.79-1.41). Mycobacterium tuberculosis complex was isolated from 31 of 82 HIV-positive patients (37.8%) with available mycobacterial blood culture results. Patients in Simplified Severe Sepsis Protocol received significantly more IV fluids in the first 6 hours (2.7 L vs 1.7 L, p = 0.002). The study was stopped early because of high mortality rate among patients with hypoxemic respiratory failure in the intervention arm (8/8, 100%) compared with the control arm (7/10, 70%; relative risk, 1.43; 95% CI, 0.95-2.14). CONCLUSION:Factors other than tissue hypoperfusion probably account for much of the end-organ dysfunction in African patients with severe sepsis. Studies of fluid-based interventions should utilize inclusion criteria to accurately capture patients with hypovolemia and tissue hypoperfusion who are most likely to benefit from fluids. Exclusion of patients with severe respiratory distress should be considered when ventilatory support is not readily available.

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Simplified 3D protocol capable of generating early cortical neuroepithelium.

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Simplified 3D protocol capable of generating early cortical neuroepithelium.

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