Yu Z, Gauthier P, Tran QT, et al. Differential properties of human being ALP+ periodontal ligament stem cells vs their ALP- counterparts. and GMSCs (GMSCs from A and B two donors). Bottom panel: Non-OSCs including BMMSCs, FFs and DFs. Genes tested included neural genes nestin, III-tubulin, NFM, Nav1.6 and CNPase. Level pub: DPSCs, all 50 m; SCAP, all 100 m; GMSCs and BMMSCs, all 50 m; DF, nestin, 100 Wisp1 m; NFM, 200 m; Nav1.6, 50 m. NIHMS1509703-supplement-Supp_Fig_3.tif (83M) GUID:?2753DB42-DD8C-42A5-AFB0-C4FD1B1178BF Supp Fig 4: Neurosphere formation and differentiation of DPSCs (Method-1). Upper panel: (A, B) DPSC-derived neurospheres after 3 days cultured in NDM-1 stage-1. (C) Neural differentiation of DPSC-derived neurosphere on day time 2 (NDM-1 stage-2). (D) After 6 days of neural differentiation (NDM-1 stage-2), fibroblast-like cells became confluent in the central part of neurospheres. Lower panel: A few cells with long processes could be seen in the peripheral part of neurospheres, while the cell body were not spheroidal. Immunofluorescence staining showing III-tubulin-positive cells (reddish). DAPI: nuclear stain. Mouse IgG serves as the bad control. Scale pub: all the level bars are 100 m, except in (D), 500 m. NIHMS1509703-supplement-Supp_Fig_4.png (2.1M) GUID:?08652DDC-E77B-4017-9304-AD869E5CB400 Supplementary Fig 5: Neurogenesis of human being DPSC neurospheres on low-attachment and adherent plates under low and normal oxygen conditions (Method-2). (A) Formation of main (72 h) DPSC neurospheres under normoxic and hypoxic conditions at stage-1 (non-adherent images on remaining). Representative images of neurospheres cultured on adherent plates after 7 days (middle) and 12 days (right) with neurodifferentiation medium (stage-2). Related cell morphological characteristics are observed in both normoxic and hypoxic plates. Scale pub 100m. (B) Immunofluorescence analysis of neural markers. Cultured DPSC neurospheres were induced with neurodifferentiation press for 12 days under normoxic or hypoxic conditions. DAPI: nuclear stain; III-tubulin: neuronal-specific marker (green); GFAP: astrocyte-specific marker (reddish); Merged image with BRL-54443 all three fields. Scale bars: 100 m. NIHMS1509703-supplement-Supp_Fig_5.png (5.0M) GUID:?DE662A9D-3C99-48F4-B7F8-EA5A925FB0DA Supp Fig 6: Neurosphere-mediated neuronogenesis BRL-54443 (Method-3) of human being GMSCs. Neurospheres created under neural induction stage for 6C8 days as the spheres improved in size over time (stage-1). Representative images of late phase (day time 6). After which, spheres were seeded onto poly-l-ornithine/laminin coated glass coverslips or tradition wells and stimulated under neural maturation medium for ~4 weeks (stage-2). The cells gradually showed spherical cell body and axon-like extensions over time. Representative images showing early phase (day time 4) and late phase (day time 28). Control: non-stimulated. Level bars: Top panel, 500 m (remaining 2 images,) 100 m (right 2 images). Bottom panel, 100 m (remaining 2 images), 50 BRL-54443 m (right 2 images). NIHMS1509703-supplement-Supp_Fig_6.png (3.9M) GUID:?1270E2F0-80EF-4A87-9B1C-A5CCC9B1C4C3 Supp Materials Methods. NIHMS1509703-supplement-Supp_Materials_Methods.docx (39K) GUID:?C3EC3F59-3399-4113-AE72-5A302E6237D2 Supp Table 1. NIHMS1509703-supplement-Supp_Table_1.doc (54K) GUID:?77479A5F-F9EF-4917-A4E5-F8C322DBE14B Supp Table 2. NIHMS1509703-supplement-Supp_Table_2.docx (22K) GUID:?CE697DFE-5BA6-42D7-8530-1AB519C46B0B Supp Table 3. NIHMS1509703-supplement-Supp_Table_3.DOCX (16K) GUID:?5382AA05-5B05-419F-A0AD-B862F532C4E2 Supp Table 4. NIHMS1509703-supplement-Supp_Table_4.docx (15K) GUID:?52A93016-5B33-4DDF-94BB-599F88FF0237 Supp Table 5. NIHMS1509703-supplement-Supp_Table_5.docx (27K) GUID:?3DF95AAC-8731-4AF1-BDFE-1B9C4D6EB6C9 Video for Supp Fig 7: Neurosphere-mediated neuronogenesis (Method-3) of GMSCs. After stage-1 neurosphere step, the spheres were seeded onto wells of a 24-well plate under stage-2 NMM. Determined locations of the attached spheres were imaged consecutively starting at day time 1 throughout the maturation period. The images were combined into a video format. Two locations each contains a single cell were traced (reddish and green package) backward from day time 28 to day time 1. From day time 28 to 17, the same cell was identified and traced. Days before the 17th day time, the same cells were not easily recognized due to migration or crowdedness of cells. NIHMS1509703-supplement-Video_for_Supp_Fig_7.mp4 (19M) GUID:?146EDAB5-6AEB-4CD0-A389-B17EFC176FB5 Abstract The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) like a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for his or her neurogenic potential compared to non-OSCs and used numerous neurogenic induction methods. OSCs including dental care pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere- mediated or neurosphere-mediated methods to guide them.