A comparative study of growth kinetics, in vitro differentiation potential and molecular characterization of fetal adnexa derived caprine mesenchymal stem cells

Abstract

<p><b>Fig-1 </b>Culture and expansion of caprine fetal adnexa derived MSCs observed under bright field microscope. a) Amniotic Fluid, b) Amniotic Sac, c) Wharton’s Jelly and d) Cord Blood monolayer represents the primary culture. (e),(f),(g) and (h) represents the 1^st passage of respective cell lineages. (i),(j),(k) and (l) represents the 3^rd passage of respective cell lines. (Magnification-10X).</p><p><b>Fig-2(A) </b>Calibrated growth curve plotted for caprine fetal adnexa derived MSCs at P3<b>. (B) </b>Population doubling time for caprine fetal adnexa derived MSCs at P3<b>. (C) </b>CFU assay for caprine fetal adnexa derived MSCs at P3. Data represents the mean±SEM. Means bearing different superscripts differ significantly (P<0.05) in different cell lines. </p><p><b>Fig-3 </b>Alkaline Phosphatase staining of caprine fetal adnexa MSCs.a) Amniotic Fluid, b) Amniotic Sac, c) Wharton’s Jelly and d) Cord Blood monolayer under bright field. e) Amniotic Fluid, f) Amniotic Sac, g) Wharton’s jelly and h) Cord Blood monolayer positive for AP stain (Magnification-20X).</p><p><b>Fig-4 </b>Multi-lineage differentiation of caprine fetal adnexa MSCs. a) Amniotic Fluid, b) Amniotic Sac, c) Wharton’s Jelly and d) Cord Blood monolayer subjected to chondrogenic differentiation. e) Amniotic Fluid, f) Amniotic Sac, g) Wharton’s Jelly and h) Cord Blood monolayer subjected to osteogenic differentiation. i) Amniotic Fluid, j) Amniotic Sac, k) Wharton’s Jelly and l) Cord Blood monolayer subjected to adipogenic differentiation (Magnification-40X).</p><p><b>Fig-5 </b>Immunocytochemical localization of cell surface antigens in caprine fetal adnexa MSCs. a) Amniotic Fluid, b) Amniotic Sac, c) Wharton’s Jelly and d) Cord Blood monolayers stained with primary antibodies directed against CD90. e) Amniotic Fluid, f) Amniotic Sac, g) Wharton’s Jelly and h) Cord Blood monolayer stained with primary antibodies directed against CD105. i) Amniotic Fluid, j) Amniotic Sac, k) Wharton’s Jelly and l) Cord Blood monolayer stained with primary antibodies directed against CD73 (Magnification-20X).</p><p><b>Fig-6</b> Immunocytochemical localization of pluripotency markers in caprine fetal adnexa MSCs. a) Amniotic Fluid, b) Amniotic Sac, c) Wharton’s jelly and d) Cord Blood monolayers stained with primary antibodies directed against Sox2. e) Amniotic Fluid, f) Amniotic Sac, g) Wharton’s Jelly and h) Cord Blood monolayer stained with primary antibodies directed against Nanog. i) Amniotic Fluid, j) Amniotic Sac, k) Wharton’s Jelly and l) Cord Blood monolayer stained with primary antibodies directed against Oct4 (Magnification-20X). </p><p><b>Fig-7</b> Immunocytochemical localization of pluripotency markers in caprine fetal adnexa MSCs. a) Amniotic Fluid, b) Amniotic Sac, c) Wharton’s Jelly and d) Cord Blood monolayers stained with primary antibodies directed against KLF. e) Amniotic Fluid, f) Amniotic Sac, g) Wharton’s Jelly and h) Cord Blood monolayer stained with primary antibodies directed against FoxD3. i) Amniotic Fluid, j) Amniotic Sac, k) Wharton’s Jelly and l) Cord Blood monolayer stained with primary antibodies directed against cMyc (Magnification-20X).</p><p><b>Fig-8 </b>Overlay histogram of flow cytometric analysis of cell surface markers CD73, CD105, CD34. The empty histogram represents the analysis with mAbs in MSCs.  </p><p><b>Fig-9 </b>Overlay histogram of flow cytometric analysis of pluripotency markers Oct4, Sox2 and Nanog. The empty histogram represents the analysis with mAbs in MSCs. </p><p><b>Fig-10 (A) </b>Gel electrophoresis picture of cell surface antigens (CD73, CD90 and CD105) and negative marker (CD34). Lane M: 50 bp Ladder; Lane 1: cAF-MSCs; Lane 2: cAS-MSCs; Lane 3: cWJ-MSCs; Lane 4: cCB-MSCs.<b> (B) </b>Gel electrophoresis picture of pluripotency markers (Oct4, Sox2, Nanog, KLF, cMyc) and endogenous control (RPS15A). Lane M: 50 bp Ladder; Lane 1: cAF MSCs; Lane 2: cAS MSCs; Lane 3: cWJ MSCs; Lane 4: cCB MSCs.</p><p><b>Fig-11 </b>Relative mRNA expression of cell surface antigens (CD73, CD90 and CD105) in caprine fetal adnexa MSCs at P3. Data represents the mean±SEM. Means bearing different superscripts differ significantly (P<0.05) in different cell lines.</p><p><b>Fig-12 </b>Relative mRNA expression of pluripotency markers(Oct4, Sox2, Nanog, KLF, cMyc) in caprine fetal adnexa MSCs at P3. Data represents the mean±SEM. Means bearing different superscripts differ significantly (P<0.05) in different cell lines. </p><p> </p><p> </p><p><br></p