Proliferation rate and gene expression of IDPSCs after cultivation in three distinct culture media. A

<p>) Proliferation curve of LP before cryopreservation; <b>B</b>) Proliferation curve of LP after cryopreservation. <b>C</b>) Gene expression of LP after cryopreservation.</p

Scaling-up of IDPSCs.

<p>Horizontally, the process of DP <i>in vitro</i> plating (Day 0, P0) followed by DP adherence and cells outgrowth (Day 3–4). This process is followed by enzymatic treatment (P1) of the cells and formation of multiple colonies (CFU-f - <u>C</u>olony <u>F</u>orming <u>U</u>nits-<u>f</u>ibroblast). After 5 days, enzymatic treatment was performed to harvest multicolony-derived IDPSCs (P2) population. Next, <i>in vitro</i> expansion of IDPSCs (P3) has been performed. Upper numbers represent approximate quantity of harvested IDPSCs in each passage. Vertically, the same process is shown, albeit after multiple DP mechanical transfer.</p

Dental pulp and IDPSCs.

<p><b>A</b>) Highly vascularized (black arrows) DP just after extraction. <b>B</b>) Explant culture of DP with outgrowing IDPSCs. <b>C</b>) Culture of IDPSCs at 1^st passage. <b>D</b>) IDPSCs showing ES-like cells morphology with a large nucleus. <b>E</b>) IDPSCs showing MSC-like morphology with several pseudopodes. <b>F</b>) IDPSCs showing uniform morphology resembling ES cells and MSCs. <b>G</b>) Karyotype of IDPSCs (LP): chromosomes in pairs, ordered by size and position did not reveal any numerical changes in chromosome number; G-banding analysis. A–C, G) Light Microscopy; D–F) Transmission Electron Microscopy; A = 20X, G = 63X; Scale bars: B = 20 µm; C, F = 10 µm; D, E = 3 µm.</p

Cell number (x10³) of IDPSCs (passage range P3–P7) cultured in different growth media before and after cryopreservation.

a<p>Values are mean±standard deviation, n = 5. Means followed by the same letter are not statistically different (Tukey, p>0.05).</p

Number of IDPSCs cultured in three different growth media and at different passages before and after cryopreservation.

a<p>DMEM-LG did not support cell growth.</p

Characterization of EP and LP of IDPSCs.

<p><b>A1–F1)</b> Flow cytometry showing EP of IDPSCs, which highly expressed such markers as SH2/CD105 (A1); SH3/CD73 (B1); nestin (C1); vimentin (D1); fibronectin (E1). <b>F1)</b> Low expression of Oct3/4 in EP; <b>A2–F2)</b> Flow cytometry showing LP of IDPSCs, which expressed same markers as EP. <b>F2)</b> Higher expression of Oct3/4 in LP, than in F1. <b>A3–F3)</b> Immunofluorescence of LP of IDPSCs using same markers as in (A2–E2). <b>F3)</b> Nuclear localization of Oct3/4 can be observed. A3–F3) Epi-fluorescence, nuclei stained with DAPI (blue). Scale bars: A3, B3, E3, F3 = 5 µm; C3, D3 = 10 µm.</p

<i>In vitro</i> differentiation potential of IDPSCs.

<p><b>A–C</b>) Chondrogenic differentiation. <b>A</b>) Pellet culture: collagen fibers intensively stained by Massoǹs thrichrome. Inset: same as in (A) high magnification. <b>B</b>) The proteoglycans presence was revealed by Toloudine blue staining. <b>C</b>) RT-PCR shows the expression of COMP gene in EP and LP of IDPSCs. Housekeeping gene GAPDH is used as control. <b>D–O</b>) Myogenic differentiation. <b>D, E</b>) Morphological aspect showing stages of muscle fibers formation. <b>F</b>) Nuclear expression of MyoD1 protein in LP of IDPSCs-derived myocyte-like cells. <b>G</b>) Myosac composed by MyoD1 positive cells. <b>H, I</b>) Titin protein expression in LP of IDPSCs-derived myotubes. <b>J</b>) Expression of troponin I in Z-bands of myofibers. <b>K</b>) Myosin protein expression. <b>L</b>) Very small, satellite-like cells, showing positive myosin immunostaining. <b>M</b>) Binuclear cell positive for alpha-actinin (spot-like labeling). <b>N)</b> Fused myotubes, which deferentially express alpha-actinin protein. <b>O</b>) RT-PCR shows the expression of MyoD1 and ACTB genes in EP and LP of IDPSCs. Housekeeping gene GAPDH is used as control. A, B, D, E) Light Microscopy; F-N) Epi-fluorescence, nuclei stained with DAPI (blue). Scale bars: A = 200 µm; B = 20 µm; D = 50 µm; E, N = 10 µm; F–M = 5 µm.</p

Functional classification of transcripts that originated from upregulated contigs.

<p>Functional classification of transcripts that originated from upregulated contigs.</p

qRT-PCR of infestin.

<p>Adult insects infected with <i>T. cruzi</i> and uninfected <i>T. infestans</i> were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired <i>t</i> test was performed for statistical analysis.</p

Functional classification of transcripts from all clusters.

<p>Functional classification of transcripts from all clusters.</p