In addition we also injected the neurospheres

In addition, we also injected the neurospheres into SCID mouse brains. The confocal images showed that the AdSTEP-fragmented neurospheres had abundant PAX6-positive Fmoc-O-Phospho-Tyr-OH manufacturer in the cerebral cortex (Fig. 6C) that contained larger NTTR structures, similar to those observed in vitro. These results suggested that the engrafted neurospheres were further differentiated to neural precursor cells, which further contributed to neurogenesis (Fig. 6D). Therefore, our AdSTEP neurospheres, generated under defined culture conditions, easily integrated into the mouse brains, demonstrating a great promise for neurogenesis studies and stem cell therapy. Overall, this novel and rapid virus-free method for generating neuronal populations from neurospheres has many advantages, all of which will have a great impact on our understanding of neuronal identity after neurosphere transplantation as well as the mechanisms of disease. The following are the supplementary data related to this article.
Authors\' contributions
Acknowledgments This work was supported by the National Institutes of Environmental Health Sciences (1R15 ES019298-01A1).
Resource details Lentiviral particles were produced with hSTEMCCA, VSV-G, Gag/Pol, Tat and Rev and the first colonies emerged 12days after transduction (Fig. 1A–C). The hSTEMCCA transgene was silenced in ihFib3.2 at passage 7 (Fig. 1G). The expression of pluripotent stem cell markers was confirmed by RT-PCR (OCT4, SOX2, NANOG, REX1, KLF4, DNMT3B, TDGF, GDF3, LIN28 and NODAL) (Fig. 1H), flow cytometry (OCT4, NANOG, SOX2, SSEA4, TRA-1–60 and TRA-1–81) (Fig. 1I) and immunofluorescence (OCT4, NANOG, SOX2, cMYC, SSEA4, TRA-1–60 and LIN28) (Fig. 1J). Moreover, GTG banding revealed a normal karyotype (46, XX) (Fig. 1F). To demonstrate differentiation into the three embryonic germ layers, ihFib3.2 were aggregated to form embryoid bodies and allowed to attach to tissue culture plates (Fig. 2A–B). Spontaneous differentiation induced the transcription of the following genes: NES and TUBB3 (ectoderm), T, BMP4 and MSX1 (mesoderm), and AFP, GATA6 and SOX17 (endoderm) (Fig. 2C). In addition, the formation of the three germ layers was confirmed at the protein level by immunofluorescence, which demonstrated the expression of Nestin, Brachyury (Fig. 2D) and α-fetoprotein (Fig. 2E).
Materials and methods
Acknowledgements This work was supported by the following Brazilian Institutions: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (467337/2014-4), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E-26/110.096.2013), Ministério da Saúde (DECIT) (420092/2013-7), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Financiadora de Estudos e Projetos (FINEP) (01.10.0115.04).
Resource table:
Resource table: Resource details Up to now, human amniotic fluid stem cells have been reprogrammed without ectopic factors (Moschidou et al., 2012). Herein, we established the reprogramming of mouse AF cells through a protocol that involves the use of a non-viral method, the PB system. AF was collected from foetuses, at embryonic days 13.5, obtained from Oct4GFP mice, useful to monitor the expression of the endogenous Oct4 in reprogrammed cells. The same method has been used to reprogram AF cells from mice constitutively expressing GFP. Mouse AF cells were transfected with circular PB-tetO2-IRES-OKMS, which expresses the Yamanaka factors linked to mCherry fluorescent protein in a doxycycline-inducible manner, (Fig. 1A and (Woltjen et al., 2015)) and reverse tetracycline transactivator (rtTA) plasmid in conjunction with the PB transposase expression plasmid (mPBase) (Woltjen et al., 2009). The transfection of mouse cells was performed after 1week of in vitro expansion over a MEF feeder layer according to the protocol described in Fig. 1B, in two separate experiments. Cells were maintained in culture, without passaging, in the presence of doxycycline at the concentration of 1.5μg/ml. The first PB-tetO2-OKMS-induced colonies depicting an ES-like morphology appeared 20days after doxycycline induction. They were picked 30days post-transfection and seeded on inactivated feeder layer. After isolation, surviving clones were maintained in medium supplemented with doxycycline for a few passages until they became doxycycline-independent (3 out of 54=5% for Oct4GFP, 4 out of 30=13% for constitutive GFP) in replicate wells, as shown in Fig. 1C. The tested clones proved to be positive for the alkaline phosphatase and for the pluripotency markers Oct4, Sox2 and Nanog (Fig. 1D). The same pluripotency marker expressions were evaluated in miPS-AF-GFP (Fig. 1E). iPS cells were also tested for the expression of endogenous and exogenous pluripotency genes (Fig. 1F): as expected, all the endogenous genes Oct4, Klf4, Myc and Sox2 were activated in the iPS cell clones whereas the corresponding exogenous genes, defined as transgene (Tg), were expressed in the presence of doxycycline and down regulated or completely silenced when cultured without doxycycline. To evaluate the capacity of iPS cell clones to differentiate into cells belonging to the three germ layers, cells underwent EBs differentiation and after 21days they resulted positive for the expression of Vim/T-brachyury/αSMA (mesoderm), Afp (endoderm) and Tubb3 (ectoderm), as showed in Fig. 2A,D. We also injected iPS cells into immunocompromised mice, and after 6weeks tumour masses arose (Fig. 2B). Teratomas were processed, analysed and tissues belonging to the three germ layers were found as showed by the haematoxylin and eosin staining (Fig. 2D) and by the positivity for the differentiation markers (Fig. 2C,D). These demonstrated that miPS-AF-(Oct4GFP/GFP) possessed the ability to differentiate into cells belonging to tissues of the three germ layers.