Activated charcoal composite biomaterial promotes human embryonic stem cell differentiation toward neuronal lineage

Abstract

Transplantation of biomaterial scaffolds encasing human embryonic stem cells (hESCs) has been proposed as a clinical therapy for various neurological lesions and disorders. In light of recent developments, artificially synthesized carbon-based biomaterials such as carbon nanotubes and graphene have demonstrated feasibility in supporting stem cell attachment and differentiation. However, the applicability is significantly hampered by evidence of nanotoxic effects on multiple cell types. Thus, an emergent drive for an innovative carbonaceous biomaterial calls for a safer platform with comparable advantageous characteristics. Here, we showed for the first time, a natural coal-based activated charcoal (AC) composite biosubstrate can support and promote neuronal differentiation in hESCs. The bio-friendly AC composite biomatrices resulted in more matured neuron-like cells. Both of axonal length and density were at least twice as long and abundant, respectively, when compared with control groups. A functional assay demonstrated that the derived neuron-like cells responded to depolarization-dependent synaptic recycling and may contain active synapses. In addition, the AC composite substrate can serve to concentrate growth factors and cell adhesion proteins, further encouraging attachment and hESC differentiation. Moreover, the AC composite biomaterial can potentially be economically manufactured as implantable three-dimensional bioscaffolds, facilitating the regeneration of damaged neural and other tissues.

Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)

“…[29][30][31][32] The standard 20% KO serum replacement medium contained 20% KO serum replacement (Invitrogen), 1% nonessential amino acids (Invitrogen), 1 mM L-glutamine (Invitrogen), Dulbecco's modified Eagle's medium (DMEM/ F12; Invitrogen), 0.1 mM b-mercaptonethanol (Sigma-Aldrich), and 4 ng/mL FGF-2 (Sigma-Aldrich). The medium was changed every day and hESCs were passed every 7 days.…”

“…To allow attachment, EBs were transferred to wells coated with PLO and Laminin 30,32 and subsequently incubated with N2 medium supplement consisting of DMEM/F12, nonessential amino acids, sodium pyruvate (Invitrogen), N2 supplement (Invitrogen), 0.1 mM b-mercaptonethanol (SigmaAldrich), and FGF-2 (8 ng/mL). For experimental purpose, EBs were treated with N2 medium containing iron (III) chloride at various concentrations (100 nM, 500 nM, and 1 mM) (Sigma-Aldrich) for a maximum of 14 days.…”

“…Axonal length was quantified in accord to our protocol published previously. 30 In brief, more than 20 fluorescently stained axons were measured at random from each of *10 EBs. Axons were measured (in pixels using NIS-Elements software) from the tip of axons to the end and averaged.…”

“…Axonal density was quantified in accordance with our previously published protocol. 30 In short, axonal density was assessed by quantifying the total fluorescence intensity of axons using random placement of more than 30 defined regions of interest. Axonal density was measured from more than 10 EBs in pixels and analyzed with NIS-ELEMENTS software.…”

“…30 In brief, cells were carefully rinsed with DPBS, loaded with FM1-43 dye (2 mM) (k Ex = 510 nm and k Em = 626) (Invitrogen) for 5 min, and were carefully rinsed again. Exocytosis of synaptic vesicles was triggered by 50 mM glutamic acid, 200 mM acetylcholine (ACh), or depolarizing solution (high K + ) (in mM: 20.9 NaCl, 100 KCl, 1.2 MgCl 2 , 1.2 NaH 2 PO 4 , 1.2 Na 2 SO 4 , 2.5 CaCl 2 , 25 NaHCO 3 , and 10 glucose, pH 7.3; Sigma-Aldrich).…”