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

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

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

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

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

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

<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

Smooth-muscle-α-actin (SMA) expression before (at day 0) and after (at day 7) eAT-MSCs intrauterine transplantation.

<p>A, B) At day 0, SMA expression was observed in uterine glands (white arrowhead) and in periglandular fibroblasts (black arrow). A1) At day 7, SMA showed no signs of expression. B) Pattern of SMA expression is similar to A and B. LM. Scale bars: A = 25 µm; A1, B, B1 = 50 µm.</p

Proliferation rate analyzed by Ki-67 antigen expression.

<p>Proliferation rate analyzed by Ki-67 antigen expression.</p

Histological analysis of alterations in mares’ endometrium following eAT-MSCs intrauterine transplantation.

<p>A–D3) Mares which received the cells. DE–F3) Control mares. A–F) Morphology of endometrial surface prior eAT-MSCs intrauterine cells transplantation. A1–D1) Day 7 after eAT-MSCs intrauterine transplantation. A2–D2) Same as in (A1–D1) at day 21. A3–D3) Same as in (A1–D1) at day 60. E1–F3) Respective controls. LM. Scale bars: A–F3 = 50 µm.</p