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Despite the lack of any bioactive domains in the substrate, we found that iPSCs encapsulated in RADA16-I prior to initiating neuronal induction led to the development of a population of iN cells with robust neurite outgrowth


Sep 27, 2021

Despite the lack of any bioactive domains in the substrate, we found that iPSCs encapsulated in RADA16-I prior to initiating neuronal induction led to the development of a population of iN cells with robust neurite outgrowth. than traditional 2-D culture substrates. A promising biomaterials platform for the support of 3-D neuronal cultures involves the design of self-assembling peptides, which are fabricated from natural amino acids and can undergo spontaneous assembly into nanofiber scaffolds when exposed to physiological media or salt solutions.9,10 One example of these self-assembling peptide nanofiber scaffolds (SAPNS) is RADA16-I, formed from alternating sequences of the amino acids arginine LOXL2-IN-1 HCl (R), alanine (A), and aspartic acid (D). When exposed to millimolar amounts of monovalent cations, the peptides undergo self-assembly to form stable attachment and growth of a wide variety of mammalian cells, LOXL2-IN-1 HCl particularly neural stem/progenitor cells.13C17 disease modeling, and drug screening.20C22 Mature, functional neuronal cells can be directly generated from iPSCs via transfection with one or more key transcription factors.23,24 Additionally, iPSCs are highly expandable and give us the ability to generate large numbers of neurons, whereas an extremely limited amount of induced neurons can be generated by directly reprogramming fibroblasts. This approach is advantageous due to rapid neuronal conversion (neurons are generated in approximately 2 weeks) and the ability to generate patient-specific neurons and specific subtypes of neurons. In comparison, traditional methods of differentiation can take months to generate neurons. In this study, we investigated the role of self-assembled peptide nanofibrous scaffolds (SAPNS) based on the RADA16-I peptide as a 3-D culture environment for the reprogramming and functional maturation of neurons derived from iPSCs. Neurons were generated via the lentiviral-mediated transfection of human iPSCs, inducing these cells to express the single neuronal transcription factor NeuroD1 and undergo subsequent neuronal conversion. Further, we fabricated monodisperse RADA16-I microspheres using a custom-made microfluidic T-junction device and evaluated these microspheres as a vehicle for the minimally invasive delivery of iPSC-derived neurons into a mouse brain. This is the first report of using SAPNS for the culture and transplantation of human neurons. MATERIALS AND METHODS Lentiviral Infection of HJ1 iPSCs. Human iPSCs (HFF1-iPSC) were obtained from the Rutgers University Cell and DNA Repository (RUCDR). iPSCs were infected with lentiviruses as previously described.24,25 Briefly, iPSCs were treated with Accutase (Life Technologies) and plated as single cells in mTeSR-1 medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 5 and gene expression. The medium was replaced every 2C3 days for the duration of the experiments. Encapsulation of Cells within Bulk RADA16-I Hydrogels. RADA16-I, a self-assembling peptide with the amino acid sequence [COCH3]-RADARADARADARADA-[CONH2] (PuraMatrix), was kindly provided by 3-D Matrix, Inc. (Waltham, MA) at a concentration of 1% (w/v). RADA16-I (diluted to 0.5% in 10% sucrose) was sonicated in a bath sonicator for 30 min and LOXL2-IN-1 HCl vortexed prior to gelation. To encapsulate cells within RADA16-I, iPSC-RNs were treated with Accutase and washed once in 10% sucrose prior to resuspending in fresh 10% sucrose at a concentration of 10 106 or 20 106 cells/mL. The cell suspension and RADA16-I peptide were mixed by gentle pipetting in a 1:1 ratio for LOXL2-IN-1 HCl a final concentration of 5 106 or 10 106 cells in 0.25% RADA16-I, prior to transferring the mixture onto a UV-sterilized coverslip in a 24 well TCPS plate (100 for 30 s to collect the microspheres, and washed once with DMEM/F12 medium. After centrifuging again at 200for 1 min, the DMEM/F12 supernatant was removed, and the microspheres were transferred to mTeSR-1 medium containing 5 Transplantation. All animal experiments were carried out according to the Rutgers University Policy on Animal Welfare and were approved by the Institutional Animal Care and Use Committee (IACUC) at Rutgers University Robert Wood Johnson Medical School. null mice (20C35g; Jackson Laboratory) were anesthetized with isoflurane (induction at 4% and maintained at 0.5C1% inhalation). At day 8 after initiating neuronal induction, GFP+ iN cells.