Schematic figure of comparative mechanism of mDA neuron differentiation between mESC and P-iPSC.

<p>The SFRP1 level is the highest in undifferentiated cells. As differentiation begins, SFRP1 expression was decreased by specific methylation at 17^th, 18^th, 21^st, and 22^nd CpGs of its promoter. Epigenetically repressed SFRP1 gene fails to antagonize Wnt5a, which enables to augment Wnt5a signal transduction. Increased level of Wnt5a binds to the Frizzled (Fz) receptor and propagates dopamine neuron differentiating-signals by stimulating Lmx1a, b, Nurr1, Pitx3 and finally inducing tyrosine hydroxylase enzyme to specify neural precursor cells to mDA neurons.</p

In vitro characterization of pluripotency of mESCs and P-iPSCs and schematic overview of experimental design for mDA neuronal differentiation.

<p>(A) Confocal images of mESCs and P-iPSCs to show pluripotency markers, Oct-4 and SSEA1 within cell colonies. Nuclei were stained with Sytox blue. Oct-4, and SSEA1 were expressed in nucleus or membrane respectively. Scale bars  = 20 μm. (B) DA neuronal differentiation is composed of five stages. Each stage showed distinct morphological changes in stem cells. The aggregated form of EBs was attached on gelatin-coated dish for 7 days. During this step, cells were transformed into tightly-packed epithelial morphology in ITSFn media. Propagation of neuronal precursor cells begins when cultured in N3 media for 3 to 5 days. Lastly, terminal differentiation into mDA neuron begins after culturing them in N3 media for 8 to 10 days in the absence of bFGF and addition of ascorbic acid in N3 media for 8∼10 days.</p

Transplantation of neuronal precursor cells of mESCs and P-iPSCs In vivo Parkison's disease model of rats.

<p>(A) Nano particle-labeled injected cells are shown as red which were observed in the graft region. Majority of TH-positive cells (green) were also Tuj1 (white),(B) VMAT2 (white) and (C) Pitx3-positive cells (white). Scale bars  = 20 μm. (D) Quantitative analysis of cells counted against nucleus marker, DAPI in three random fields revealed that higher number of cells existed with neuronal-positive signals in P-iPSCs when compared to mESCs injected groups. (E) Total apomorphine-induced rotation numbers (0.5 mg/kg) were counted at two weeks after cell injection. mESC (n = 7) and P-iPSC injected groups (n = 7) showed the improved symptom compared to PBS injected groups (n = 4), while average rotation score of P-iPSC group was slightly decreased compared to mESC injected group. Each value depicts mean ± SEM of number of rotation. PBS group vs mES group (*P<0.005), PBS group vs P-iPS group (*P<0.005).</p

Comparison of Wnt5a expression between mESCs and P-iPSCs during neuronal differentiation.

<p>(A) Gene expression of Wnt5a at stage 1,3 and 5 during differentiation between mES and P-iPS cells was confirmed by real-time PCR. While Wnt5a mRNA levels in mESCs were gradually increased, the signals in P-iPSCs were rapidly increased from S3, resulting in the higher Wnt5a expression in S5 of P-iPSCs than mESCs. (B) Immunofluorescent staining of TH and Wnt5a between mES and P-iPS at stage 5. Immunofluorescence data reveal more Wnt5a–positive cells leads to have higher TH–positive protein expressions in P-iPSCs than in mESCs. Scale bars  = 20 μm.</p

Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials

<div><p>Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1⁺ (Sca-1⁺/PDGFRα⁺ and Sca-1⁺/PDGFRα⁻) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1⁻ (Sca-1⁻/PDGFRα⁺ and Sca-1⁻/PDGFRα⁻) progenitor cells. Among Sca-1⁺ progenitor populations, Sca-1⁺/PDGFRα⁻ cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1⁺/PDGFRα⁺ cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1⁺/PDGFRα⁻ cells preferentially differentiated into osteoclast-like cells. Sca-1⁺ progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1⁺ calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1⁺ progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1⁺/PDGFRα⁻ cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1⁺/PDGFRα⁻ cells into Apoe^−/− mice increased the severity of calcified atherosclerotic plaques. However, Sca-1⁺/PDGFRα⁻ cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1⁺/PDGFRα⁻ cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.</p> </div

P-iPSCs-derived EBs have more potent migration capacity compared mESCs-derived EBs.

<p>(A, B) Microscopic image of mESCs and P-iPSCs in hanging drop culture for 4 days. Both mESCs and P-iPSCs formed similar spherical-shaped mass. (C–E) Sprouting cells from EBs were observed and the morphologically appearing largest migration distance from the outer region of aggregated EB to farthest region within the migration zone (indicated by black dashed-line and double-ended red arrow) was measured after 24 hours post-attachment. Data are presented as mean ± SEM. The symbol * denotes high statistical significance (P<0.05), mES vs P-iPS, All values are representative of three independent experiments, Scale bars  = 20 μm.</p

Comparison expression analysis of mouse DA neuronal specific markers between mESCs and P-iPSCs during neuronal differentiation.

<p>(A) Gene expression of previously reported mDA neuronal specific markers was confirmed by quantitative RT-PCR during neuronal differentiation. Following mRNA expression represents relative gene expression at stage 5 compared to stage 1. Most of gene expression of markers was relatively stronger in P-iPSCs than mESCs. These experiments were repeated three times. (B) Representative immunofluorescence data of mESCs and P-iPSCs at stage 5. Stronger TH-positive cell signals and more numbers of double-positive cells (TH/Tuj1 or/Nurr1 or/Pitx3 or/VMAT2) were observed in P-iPSCs than mESCs. Scale bars  = 20 μm. (C) Total cell numbers of mDA specific marker positive were counted in randomized fields (n = 10) out of 5,000cells, revealing that more abundant number of cells was counted in P-iPSCs. Data are presented as mean ± SEM (* P<0.05). All values are representative of 3 independent experiments. (D) The percentage of TH-positive cell number divided by neuronal marker-positive cell numbers was shown in bar graph. The bar graph shows the yield of terminal differentiation marker, TH-positive neurons from neural precursor cells. Data are presented as mean ± SEM (* P<0.05; ** P<0.01). All values are representative of three independent experiments. (E) Quantification of TH-positive cells between two different types of mESC and P-iPSC by FACS. C57-mESC is derived from C57BL/6 mouse strain. E14-mESC is derived from 129/Ola mice. Skin fibroblast (sFB)-derived P-iPSC is primarily cultured and generated from dermis of FVB mice. Cardiac fibroblast (cFB)-derived P-iPSC is originally obtained from C57BL/6 mice heart. Each group of cells at stage 5 mDA differentiation was harvested and expression of TH was analyzed in a quantitative manner.</p

Expression level of SFRP and methylation status of its promoter during differentiation stages of mESCs and P-iPSCs.

<p>(A) mRNA expression of SFRP1 at each differentiation stages was analyzed in both mES and P-iPS cells during differentiation. The SFRP1 gene expression in three representative stages of cells was down-regulated as cells become terminally differentiated. Lower expression levels of SFRP1 gene was maintained from S3 in P-iPSCs than mESCs. Each gene expression was normalized to that of GAPDH expressions and presented relative to the respective value of the S1 mES levels. (n = 3) (B) Bisulfite sequencing of sFRP1 promoter of mES and P-iPS cells during stage 3 differentiation. Out of 42 CpGs, hypermethylation at 17^th, 18^th, 21^st, and 22^ndCpGs of SFRP1 promoter was observed between mES and P-iPSCs.</p

Calcifying progenitor cells induce calcification in atherosclerotic plaques of Apoe^−/− mice.

<p>(A) Calcium accumulation levels in mouse arteries according to diet. *<i>P</i><0.005 versus C57 fed a normal diet (<i>n</i> = 10 per group). (B) Experimental timeline. (C) Calcium accumulation levels in the aortas of Apoe^−/− mice according to BM cell type injected and PPARγ activation. *<i>P</i><0.001 versus PBS-treated group. ‡<i>P</i><0.001 versus mice injected with BM Sca-1⁺/PDGFRα⁻ cells. (D) Atherosclerotic plaque with MT staining. Bars: 200 µm. (E) Calcification induction in atherosclerotic plaques was detected by von Kossa staining. Arrows indicate calcified areas. Bars: 200 µm. (F) Atherosclerotic calcification plaque immunostained with osteocalcin, an osteoblastic marker, and RANK, an osteoclastic marker, to determine the fates of infiltrating BM-derived calcifying progenitor cells in the presence/absence of PPARγ activation. Bars: 20 µm. (G) Image enlargement showing osteocalcin and RANK immunostaining. Bars: 20 µm. (H) Osteoblastic, osteoclastic, and bidirectional cell counts in the artery. GFP⁺Sca-1⁺/PDGFRα⁺ cells primarily expressed osteoblast markers. GFP⁺Sca-1⁺/PDGFRα⁻ cells mainly differentiated into osteoblast-like cells, but some infiltrating cells differentiated into bidirectional cells. When PPARγ was activated, differentiating GFP⁺Sca-1⁺/PDGFRα⁻ cells shifted from osteoblast-like to bidirectional cells or osteoclasts (<i>n</i> = 10 per group). *<i>P</i><0.005 compared to mice injected with BM-derived Sca-1⁺/PDGFRα⁻ cells. P, PPARγ agonist.</p

Vessel resident calcifying progenitor cells are mesenchymal but not hematopoietic.

<p>(A) FACS of arterial cells from GFP–BMT Apoe^−/− mice. GFP⁺ cells were negative for Lineage antibody cocktail targets. GFP⁺Lin⁻Sca-1⁺/PDGFRα⁺ or GFP⁺Lin⁻Sca-1⁺/PDGFRα⁻ cells expressed CD29 or CD106 (<i>n</i> = 10, performed in triplicate). (B) Schematic of osteoblast, adipocyte, and chondrocyte inductions from calcifying progenitor cells. Cells were stained with Alizarin Red S after 14 d of differentiation; bars: 1 mm. Oil Red O staining 28 d after differentiation; bars: 50 µm. Safranin O staining 28 d after differentiation; bars: 50 µm. (C) Adipocyte-related genes and chondrocyte-related genes were upregulated in Sca-1⁺ cells (Sca-1⁺/PDGFRα⁺, Sca-1⁺/PDGFRα⁻) under each differentiation condition.</p