Table 1.
List of primers and their sequences or catalogue numbers used in the study.
Fig 1.
Feeder-layer independent induced pluripotent stem cells generation from HUVECs (HiPSCs).
(A) HUVECs were transduced with lentiviral vectors. Within 4 days (B) and a week (C) the endothelial cells were reduced in density but formed aggregates. (D) On day 16, immature colonies emerged. (E and F) Fully reprogrammed human embryonic stem cells (hESC)-like colonies were isolated after 21 days. Micrographs (G and I in low magnification) and (H and J in high magnification) show the morphology and quality of the colonies. Scale bars: A-F = 200 μm, G and I = 100 μm, H and J = 400 μm.
Fig 2.
Characterization of HUVEC-derived iPSCs (HiPSCs).
Gene expression was determined after 5 passages of the colonies. RT-PCR results (mRNA levels) showing endogenous embryonic (A) and endothelial (B) specific genes expressed by HUVECs and HiPSCs. Values in Figure B represent fold reduction compared to HUVECs. Data are average values of 4 HiPSC lines (N = at least 3 replicates; P<0.001). Non-detectable genes are denoted as n.d. At protein levels using immune-staining, representative micrographs show expression of embryonic markers-SSEA-4 and Oct 3/4 (C and D respectively), DAPI (E) and lack of expression of endothelial markers- PECAM (CD31) and VE-cadherin (F and G respectively) in HiPSC colonies. H is DAPI co-staining of panels F and G. Scale bar: 200 μm.
Fig 3.
Differentiation of HiPSCs to embryoid bodies (EB) and germ layers.
The ability of HiPSCs to form EB was demonstrated by dispersing colonies to single cell suspensions and placing them in aggrewell plates (A). After 24 hours, homogenous EB were formed (B). EB were differentiated to three germ layers as described in the Materials and Methods. (C and D) Germ layer formation was demonstrated by RT-PCR (C, Pax6 for ectoderm, and D, VEGFR2 for mesoderm markers respectively). (E) The endoderm marker, alpha-fetoprotein (AFP), was detected by immunofluorescence. Scale bar: 200 μm. The experiments in panels C and D were repeated 3 times; P<0.001.
Fig 4.
Differentiation of HiPSCs into neurons and astrocytes.
Representative micrographs showing that differentiated HiPSCs acquired neuronal morphology with extending neuritic processes (A) in phase contrast, and became positive for (B) MAP-2 and (C) βIII-tubulin. (D) Primary human fetal cells differentiate into neurons (green) and GFAP-positive astrocytes (red). Similarly, HiPSCs differentiated not only to neurons but also to GFAP-positive astrocytes. The micrograph in (E) shows a HiPSC-derived mixed culture of neurons (green) and GFAP immune-stained astrocytes (red). (F) A micrograph showing few mature astrocytes positive for S100B. Blue is DAPI for nuclear staining. Scale bar: 200 μm.
Fig 5.
Microarray-based gene analysis.
The graphs show cluster analysis of Row Z-score data of (A) pluripotent/embryonic, (B) endothelial, as well as (C) neuronal and glial genes expressed by HUVECs, HiPSCs, HiPSC-derived neurons (HiPSC-Ns), HFNs and samples obtained from previous publications as described in the Materials and Methods (hESCs, hESC-NSCs, hESC-SCNTs). Data are average values of 3 independent experiments.
Fig 6.
Morphological comparison of human fetal neurons (HFNs) and HiPSC-derived neurons.
Neurons were immuno-stained for MAP-2 and their morphology was manually evaluated. Micrographs showing MAP-2 positive (A) HFNs and (B) HiPSC-derived neurons. The graphs compare average (C) size of cell bodies, (D) number of neurites/cell, and (E) neurite length/cell of HFNs and HiPSC-derived neurons. P< 0.001; Scale bar: 100 μm. The experiment was repeated 3 times, and at least 50 neuronal cells were evaluated in each group.
Fig 7.
Susceptibility of HiPSC-derived neurons to inflammatory cells.
MAP-2 immuno-stained micrographs showing that HiPSC-derived neurons were either (A) treated with neuronal media, (B) co-cultured with unactivated T cells or (C) co-cultured with activated T cells. (D) shows quantification of MAP-2 positive surviving neurons.
Fig 8.
Levels of amino acids and expression of neuronal markers.
(A) HPLC results showing the levels of amino acids in both HFNs and HiPSC-derived neurons. ASP = Aspartate, GLU = glutamate, SER = Serine, GLN = glutamine, GLY = Glycine, ARG = Arginine, TAUR = Taruine, ALA = Alanine, GABA = gamma-aminobutyric acid. Data are pooled from 3 independent experiments. (B) βIII-tubulin immuno-stained total HiPSC-derived neurons. (C) Tyrosine hydroxylase (TH)-positive dopaminergic neurons. Scale bar: 200 μm.
Fig 9.
Calcium imaging and expression of functional receptors on neurons.
(A) Primary human fetal neurons (HFNs) and (B) HiPSC-derived neurons (HiPSC-Ns) were incubated with the membrane-permeant acetoxymethyl form of the fluorescent Ca2+-sensitive dye Fluo-4-AM for 1hr prior to imaging, after which the fluorescence intensity was measured. Graphs shown on left column represent responses of three HFNs, and on right responses of three HiPSC-Ns to glutamate, nicotine and acetylcholine treatments. Arrows indicate the time when the stimulants were applied. Scale bar: 100 μm. The experiments were repeated 3 times.
Fig 10.
Calcium imaging and expression of functional receptors on astrocytes.
(A) Primary human fetal astrocytes (HFAs) and (B) HiPSC-derived astrocytes (HiPSC-As) were incubated with the membrane-permeant acetoxymethyl form of the fluorescent Ca2+-sensitive dye Fluo-4-AM for 1hr prior to imaging, after which fluorescence intensity was measured. Graphs shown on left column represent responses of three HFAs and on right responses of three HiPSC-As to ATP and acetylcholine treatments. Arrows indicate the time when the stimulants were applied. Scale bar: 100 μm. The experiments were repeated 3 times.
Table 2.
Summary of cellular response to stimulants.