Induction of early neural precursors and derivation of tripotent neural stem cells from human pluripotent stem cells under xeno‐free conditions

Abstract

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however, further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal, we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference, pluripotency, and karyotype. Critically, XF transitioned H9 hESC, Shef4 hESC, and iPS6-9 retained pluripotency (Oct-4 and NANOG), proliferation (MKI67 and PCNA), and normal karyotype. Subsequently, XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors, which

Peptide‐modified, hyaluronic acid‐based hydrogels as a 3D culture platform for neural stem/progenitor cell engineering

“…Glial fibrillary acidic protein is an intermediate filament protein whose expression is associated with activated astrocytes. GFAP is upregulated in response to inflammation and in the glial scar that forms to isolate injured tissue from surrounding healthy tissue in the CNS . Levels of GFAP mRNA were significantly decreased in NS/PCs cultured in 3D, compared to 2D, in pre‐differentiation and differentiation media conditions (Fig.…”

“…Next, we evaluated expression of βIII‐tubulin (TUBB3) and doublecortin (DCX), which both indicate a neuronal or neuronal precursor phenotype (Fig. ) . A decrease in TUBB3 mRNA was observed for 2D‐cultured NS/PCs in maintenance medium at day 28 compared to those in pre‐differentiation medium at day 42 (Fig.…”

“…Despite these positive results, differentiation of NS/PCs into OPCs is inefficient. The best in vitro yields of human OPCs remain below 30% and in vivo below 25% of NS/PCs become oligodendrocytes after transplantation into the injured rodent spinal cord . While relatively few transplanted cells differentiate and engraft into the host tissue, these few are able to induce some functional improvement …”

“…Key factors in the tissue microenvironment include the extracellular matrix (ECM) and cell‐secreted soluble factors . Compared to neuron or astrocyte differentiation, oligodendrocyte differentiation of human NS/PCs has been more difficult to achieve . The majority of protocols previously described for differentiation of NS/PCs toward oligodendrocytes rely on temporal application of formulations of soluble bioactive factors, a potent method of controlling differentiation .…”

Characterization of brain cell nuclei with decondensed chromatin

“…Sox2 is expressed in most embryonic and adult NSC and its levels of expression are likely responsible for maintaining essential stem cell activities including stem cell identity, self-renewal, proliferation and neurogenesis (Ellis et al, 2004;Taranova et al, 2006;Agathocleous et al, 2009;Surzenko et al, 2013;Thiel, 2013). Although less specific for any cell type, PCNA expression parallels BrdU incorporation and is a frequently used indicator of active cell proliferation, entry into the cell cycle, and of multipotent NPCs in the brain (Marz et al, 2010;D'Amico et al, 2013;Marschallinger et al, 2014;Nguyen et al, 2014). OCT4 expression is essential to cell fate determination, neurogenesis, and pluripotency and widely accepted as prognostic of NPCs and stem cells (Shi et al, 2008;Archer et al, 2011;Luo et al, 2013;Yamada et al, 2013).…”