newborn hffs  (Kurabo industries)

Journal: Nature Communications

Article Title: Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a

doi: 10.1038/s41467-018-05209-1

Figure Lengend Snippet: Ptf1a directly converts mouse embryonic fibroblasts (MEFs) into self-renewable neurospheres. a Schematic depicting the procedure to generate neurospheres from MEFs by Ptf1a lentiviruses. MEFs were prepared from E12.5 C57BL6/J mouse embryos and infected with tet-on Ptf1a lentiviruses at day 0. Neurosphere-like structures gradually appeared in 9–14 days when cultured in N3 medium containing EGF, bFGF, and doxycycline. b Morphological changes of MEFs infected with control GFP or Ptf1a lentiviruses. Numerous cell clusters could be seen in Ptf1a virus-infected MEFs by day 6 in culture, but rarely in GFP virus-infected MEFs. From day 9 onward, many typical neurosphere-like structures formed in Ptf1a-transduced MEFs, whereas in GFP-transduced MEFs there were only few cell aggregates, which were morphologically different from typical neurospheres. c Dissociated cells from collected primary neurospheres formed secondary neurospheres in 3 days in culture and morphologically homogeneous larger neurospheres by day 7. d Quantification of Ptf1a-induced neurospheres. MEFs (4 × 10 4 ) were seeded into each well of 12-well plates, infected with Ptf1a or GFP viruses, and neurosphere-like structures in each well were then counted at day 10 and 14 following virus infection. There were significantly more neurospheres in Ptf1a-induced samples at day 10 and 14. Data are presented as mean ± SD ( n = 3). Asterisks indicate significance in unpaired two-tailed Student’s t -test: * P < 0.005, ** P < 0.0001. e EdU-labeling of Ptf1a-induced iNSCs (miNSC10) and control NSCs (SCR029). Cells were counterstained with nuclear DAPI. f Quantification of EdU-labeled cells. Higher percentage of proliferative cells was found in miNSC10 than in control SCR029 NSCs. Data are presented as mean ± SD ( n = 3). The asterisk indicates significance in unpaired two-tailed Student’s t -test: * P < 0.0001. Scale bars, 80 μm ( b ), ( e ) and 93.1 μm ( c )

Article Snippet: HEK293T cells (ATCC, CRL-3216) and human newborn foreskin fibroblasts (HFFs, from ATCC, SCRC-1041) were purchased from ATCC.

Techniques: Infection, Cell Culture, Control, Virus, Two Tailed Test, Labeling

Journal: Nature Communications

Article Title: Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a

doi: 10.1038/s41467-018-05209-1

Figure Lengend Snippet: Characterization of Ptf1a-reprogrammed iNSCs. a Neurospheres induced from MEFs by Ptf1a at day 8 and 10 were immunoreactive for Ptf1a, Sox2, Pax6, Nestin, or Olig2. Monolayered miNSC10 cells were also immunoreactive for Ptf1a, Sox2, Pax6, or Nestin. Cells were counterstained with nuclear DAPI. Scale bars, 80 μm. b qRT-PCR analysis showed that in comparison with MEFs, there was a great increase in expression of Nestin , Olig2, Pax6 , and Sox2 genes in Ptf1a-derived miNSC5, miNSC10, and miNSC12 cells, as well as in control SCR029 NSCs. Data are presented as mean ± SD ( n = 4). Asterisks indicate significance in unpaired two-tailed Student’s t -test: * P < 0.0005. c qRT-PCR assay showed that compared with mouse ESCs, there was only minimal or no expression of pluripotent factor genes Klf4 , Nanog , and Oct4 in miNSC and control NSC cells. As a pluripotent factor gene and also NSC marker gene, Sox2 exhibited high levels of expression in both miNSC and ES cells. Data are presented as mean ± SD ( n = 4). Asterisks indicate significance in unpaired two-tailed Student’s t -test: * P < 0.05, ** P < 0.005, *** P < 0.0001. d As revealed by qRT-PCR assay, expression of fibroblast cell marker genes Snai1 , Twist2 , and Col1a1 was dramatically downregulated in miNSC cells. Data are presented as mean ± SD ( n = 4). Asterisks indicate significance in unpaired two-tailed Student’s t -test: * P < 0.0001. e DNA methylation status was examined in the promoter regions of Nestin , Oct4 , and Nanog genes. Filled and empty circles represent methylated and unmethylated CpGs, respectively. Compared with MEFs, the promoter region of Nestin became hypomethylated in SCR029 and miNSC10 cells; however, the promoter regions of Oct4 and Nanog remain hypermethylated

Article Snippet: HEK293T cells (ATCC, CRL-3216) and human newborn foreskin fibroblasts (HFFs, from ATCC, SCRC-1041) were purchased from ATCC.

Techniques: Quantitative RT-PCR, Comparison, Expressing, Derivative Assay, Control, Two Tailed Test, Marker, DNA Methylation Assay, Methylation

Journal: Nature Communications

Article Title: Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a

doi: 10.1038/s41467-018-05209-1

Figure Lengend Snippet: Ptf1a reprograms human foreskin fibroblasts (HFF) into tripotent iNSCs. a , b Ectopic expression of Ptf1a in HFFs by lentiviruses induced the formation of neurospheres. c , d In the absence of doxycycline (Dox), Ptf1a-induced neurosphere cells were capable of forming neurospheres before passage 20 ( c ), but lost the capacity after passage 20 and became monolayered ( d ). e – i Ptf1a-induced human neural stem cells (hiNSCs) were highly immunoreactive for PTF1A, SOX2, PAX6, NESTIN, OLIG2, and FABP7. j – p Ptf1a-induced hiNSCs were capable of differentiating into neurons immunoreactive for TUJ1, MAP2, NEUN, TAU, or GABA, astrocytes labeled by GFAP, or oligodendrocytes marked by O1. q Neurons differentiated from hiNSCs were immunoreactive for both Tuj1 and synapsin. Cells in f – q were counterstained with nuclear DAPI. r Voltage-clamp recordings indicated fast activated and inactivated inward sodium currents as well as outward potassium currents on a differentiated neuron. s Current-clamp recordings revealed action potential responses of a differentiated neuron under current injection. t An action potential was induced after depolarization of the neuron. u Spontaneous postsynaptic currents recorded from a differentiated neuron. Scale bars, 80 μm ( a – d ) and 40 μm ( e – q )

Article Snippet: HEK293T cells (ATCC, CRL-3216) and human newborn foreskin fibroblasts (HFFs, from ATCC, SCRC-1041) were purchased from ATCC.

Techniques: Expressing, Labeling, Injection