Generation of Rat Neural Stem Cells to Produce Different Astrocyte Phenotypes

Smith, Rebecca Sherrard; Barnett, Susan C.; Lindsay, Susan L.

doi:10.1007/978-1-0716-1979-7_21

Cited by 3 publications

(3 citation statements)

References 18 publications

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“…After soyabean trypsin inhibitor/DNase treatment, cells were triturated and spun at 800 rpm for 5 min. Cells were resuspended in plating medium (50% DMEM, 25% horse serum, 25% HBSS), 2 mM L‐glutamine (Invitrogen, Paisley, UK) and plated at 150,000 cells onto a monolayer of neurosphere derived astrocytes (Smith et al, 2022 ; Sorensen et al, 2008 ), left to attach for 3 h then fed with differentiation media (DM; DMEM; 4500 mg/mL glucose), 10 ng/mL biotin, 0.5% hormone mixture (1 mg/mL apotransferrin, 20 mM putrescine, 4 μM progesterone, 6 μM selenium) formulation based on N2 mix of (Bottenstein & Sato, 1979 ) 50 nM hydrocortisone and 10 μg/mL insulin (all reagents from Sigma; Invitrogen, Paisley, UK). Cultures were maintained by replacing half of the medium three times a week.…”

Section: Methodsmentioning

confidence: 99%

Section: In Vitro De/myelinating Assaysmentioning

confidence: 99%

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Low sulfated heparan sulfate mimetic differentially affects repair in immune‐mediated and toxin‐induced experimental models of demyelination

Lindsay

McCanney

Zhan

et al. 2023

Glia

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There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes.In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model.Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.

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Section: Methodsmentioning

confidence: 99%

Section: In Vitro De/myelinating Assaysmentioning

confidence: 99%

Low sulfated heparan sulfate mimetic differentially affects repair in immune‐mediated and toxin‐induced experimental models of demyelination

Lindsay

McCanney

Zhan

et al. 2023

Glia

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“…Neurospheres (NS) were generated from the striata of 1-day-old SD rat pups using a method modified by [ 26 ]. They were differentiated into astrocytes as described in [ 9 , 14 , 27 ]. Briefly, neurospheres were triturated and plated onto 13 mm poly-L-lysine (PLL; 13 µg/mL, Sigma, Gillingham, UK)-coated coverslips (VWR, Leicestershire, England, UK) and incubated for 5–7 days in vitro (DIV) at 37 °C, 7% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Validation of Recombinant Heparan Sulphate Reagents for CNS Repair

Lindsay

Smith

Yates

et al. 2023

Biology

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Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair.

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