Gene expression of extracellular matrix components in human amniotic cells – a pilot study

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(1)DOI: 10.17772/gp/60977. Ginekol Pol. 2016, 87, 42-53.

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(3) po ł o ż n i c t wo. Gene expression of extracellular matrix components in human amniotic cells – a pilot study Ekspresja genów składników macierzy pozakomórkowej w komórkach owodni łożyska ludzkiego – badanie pilotażowe

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(6) , ! " #$ %& 1. Department of Cytophysiology, Chair of Histology and Embryology, School of Medicine in Katowice, Poland Students Scientific Society, School of Medicine in Katowice, Poland 3 Department of Molecular Biology, Chair of Molecular Biology and Genetics, School of Medicine in Katowice, Poland 4 Department of Women’s Health, School of Health Sciences, Medical University of Silesia, Katowice, Poland 2. Abstract Objectives: Identification of microenvironmental components, which significantly affect the fate of amniotic stem cells, is one of the essential conditions for their in vitro preparation for clinical applications. Control of their gene expression seems to be crucial for in vitro reconstitution of the natural amniotic microenvironment and modulation of the stem cell phenotype both, in vitro and in vivo. The aim of the study was to analyze multi-gene expressions for ECM components in human amnion cells (hACs) as compared to differentiated cells of a mesenchymal and ectodermal origin. Material and methods: hACs were isolated from normal human placentas at term. SSEA-4+ cells were visualized in vitro by immunofluorescence microscopy and counted by flow cytometry. The expressions of 84 genes were assayed by the RT-PCR Array method and analyzed by Web-based PCR Array Data Analysis Software. These expressions were compared among hACs, fibroblasts, and keratinocytes. Results: Almost 86% of the primarily cultured hACs exhibited expression of the SSEA-4 surface marker. In these cells, the expression of 7 genes for ECM structural components, 4 for adhesion proteins, 3 for integrin subunits, and 2 for metalloproteinases was strong: over 100-fold up- or down-regulated as compared to fibroblasts. These differences in gene expressions were less distinct when hAC were compared with keratinocytes.. Autor do korespondencji: Piotr Czekaj Department of Cytophysiology, Chair of Histology and Embryology Medical School of Medicine in Katowice, Medical University of Silesia, Medyków 18, 40-752 Katowice; Poland Fax : +48 32 2526574 Phone: +48 32 2088374 e-mail: pcz@sum.edu.pl.. 42. © Polskie Towarzystwo Ginekologiczne. Otrzymano: 03.03.2015 Zaakceptowano do druku: 01.04.2015. Nr 1/2016.

(7) Ginekol Pol. 2016, 87, 42-53. DOI: 10.17772/gp/60977. P R A C E. O R Y G I N A L N E po ł o ż n i c t wo. Marcin Tomsia et al. Gene expression of extracellular matrix components in human amniotic cells – a pilot study.. Conclusions: hACs exhibit highly different expression of several ECM-associated genes as compared to differentiated cells, especially of mesenchymal origin. The products of these genes, via an autoregulatory mechanism, might determine the nature of ECM-stem cell interactions, which are crucial for their stemness and potential differentiation abilities.. Key words: human amnion cells / SSEA-4 / gene expression / extracellular matrix / / basement membrane /. Streszczenie Cel pracy: Identyfikacja składników ECM istotnych dla losów owodniowych komórek macierzystych i zastosowanie ich in vitro jest jednym z kluczowych warunków odpowiedniego przygotowania tych komórek dla celowanych aplikacji klinicznych. Kontrola ekspresji genów może mieć kluczowe znaczenie dla odtworzenia warunków naturalnego mikrośrodowiska owodni in vitro oraz dla sterowania fenotypem komórek macierzystych. Celem pracy była ocena ekspresji genów składników ECM w komórkach izolowanych z owodni łożyska ludzkiego (hAC), w porównaniu z ich ekspresją w komórkach zróżnicowanych pochodzenia mezenchymatycznego i ektodermalnego. Materiał i metody: hAC izolowano z łożysk ludzkich uzyskanych po prawidłowych ciążach zakończonych terminowo cesarskim cięciem. Liczbę komórek wykazujących ekspresję antygenu SSEA-4 określano przy użyciu cytometru przepływowego. Do wizualiacji komórek SSEA-4+ w hodowli zastosowano metodę mikroskopii immunofluorescencyjnej. Badano ekspresję 84 genów metodą mikromacierzy RT-PCR i analizowano uzyskane wyniki przy użyciu oprogramowania Web-based PCR Array Data Analysis Software. Ekspresję badanych genów w hAC wyrażono jako krotność ekspresji tych samych genów oznaczonej w fibroblastach i keratynocytach. Wyniki: Około 86% hAC w hodowli pierwotnej stanowiły komórki posiadające marker SSEA-4. W komórkach tych ekspresja 7 genów składników strukturalnych ECM, 4 genów białek związanych z adhezją, 3 genów podjednostek integrynowych i 2 genów metaloproteinaz, wykazywała ponad 100-krotną różnicę w stosunku do fibroblastów. Znaczne różnice w ekspresji dotyczyły większej liczby genów niż to miało miejsce w hAC porównanymi z keratynocytami. Wnioski: hAC wykazują wysoce zróżnicowaną ekspresję niektórych genów składników ECM w stosunku do komórek zróżnicowanych, szczególnie pochodzenia mezenchymatycznego. Składniki te, pośrednio, poprzez mechanizm autoregulacyjny mogą decydować o charakterze oddziaływań ECM-komórki macierzyste, decydujących o fenotypie komórki macierzystej i potencjalnych możliwościach różnicowania.. Słowa kluczowe: komórki ludzkiej owodni / SSEA-4 / ekspresja genów / / macierz pozakomórkowa /

(8) /. Introduction Abbreviations: ECM – extracellular matrix; ESC – embryonic stem cells; hACs – human amniotic cells; hAECs – human amniotic epithelial cells; hAM-MSCs – human amniotic mesenchymal stromal cells; hESCs – human embryonic stem cells; MMP – metaloproteinases; MSCs – mesenchymal stem cells; SSEA-4 – Stage-Specific Embryonic Antigen 4. . Skróty: ECM – macierz pozakomórkowa; ESC – embrionalne komórki macierzyste; hAC – ludzkie komórki owodni; hAC – ludzkie komórki nabłonka owodni; hAM-MSC – ludzkie mezenchymalne komórki zrębowe owodni; hESC – ludzkie embrionalne komórki macierzyste; MMP – metaloproteinazy; MSC – mezenchymalne komórki macierzyste; SSEA-4 – ang. Stage-Specific Embryonic Antigen 4.. Nr 1/2016. % !

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(316) Ginekol Pol. 2016, 87, 42-53. DOI: 10.17772/gp/60977. P R A C E. O R Y G I N A L N E po ł o ż n i c t wo. Marcin Tomsia et al. Gene expression of extracellular matrix components in human amniotic cells – a pilot study.. Figure 1. Immunodetection of SSEA-4+ cells (A and B) and adequate isotype controls (C and D) in primary culture of amniotic cells. On the right side, the flow cytometric analysis of amnion-derived cells is shown. 95% of presented cells exhibit SSEA-4 expression (E). Isotype control (F) and a population of unlabeled cells (G) is also shown. FITC-conjugated anty-SSEA4 antibody – Green; F-actin stained with Rhodamine phalloidin - Red-orange; cell nuclei stained with DAPI - Blue. Scale = 20 μm.. Figure 2. Strongly up- and down-regulated ECM & adhesion genes in amniotic cells revealed by PCR Arrays. Total RNA from normal human hAEC, as well as fibroblasts and keratinocytes were characterized in technical triplicates, and the relative expression levels (< or > 100x) for each gene are plotted against each other in the Scatter Plot (outside the dashed lines). 7 genes encoding the ECM structural components, 4 genes encoding adhesion proteins, 3 genes for integrin subunits and 2 genes for metalloproteinases are up-regulated, while genes encoding matrix metallopeptidases MMP1 and MMP3 are down-regulated in hAEC in relation to fibroblasts (left plot). 3 genes encoding adhesive molecules, 2 genes encoding structural proteins and 2 genes for other ECM-associated proteins are up-regulated in hAEC in relation to keratinocytes (right plot).. Nr 1/2016. © Polskie Towarzystwo Ginekologiczne. 45.

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(579) Ginekol Pol. 2016, 87, 42-53. DOI: 10.17772/gp/60977. P R A C E. O R Y G I N A L N E po ł o ż n i c t wo. Marcin Tomsia et al. Gene expression of extracellular matrix components in human amniotic cells – a pilot study.. Table II. Expression of genes of adhesive molecules in amniotic cells in relation to differentiated cells. Genes encoding Selectin L, thrombospondin 3 and VCAM1 are strongly up-regulated in amniotic cells (shaded boxes). Thrombospondines and tenascin-C has been found to have both adhesive and anti-adhesive properties for cells. A, This gene’s average threshold cycle is relatively high (> 30) in either the control or the test sample, and is reasonably low in the other sample (< 30). These data mean that the gene’s expression is relatively low in one sample and reasonably detected in the other sample. ESC, embryonic stem cells; HA, hyaluronic acid.. . . . " #

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(581) . vs. keratinocytes. CD44. CD44 molecule (Indian blood group). Cell migration, receptor for HA. -17,70A. -18,85A. CNTN1. Contactin 1. Formation of axon connections. -1,74. -10,90A. %&. Connective tissue growth factor. Related to platelet-derived growth factor, cell proliferation and differentiation. -1,01. 4,47. CTNNA1. Catenin (cadherin-associated protein), alpha 1, 102kDa. Regulation of cadherins’ adhesion properties, cell differentiation. 1,91. 1,18. CTNNB1. Catenin (cadherin-associated protein), beta 1, 88kDa. Cell-cell interactions, component of adhesion junctions, signal transduction. 6,00. 1,87. CTNND1. Catenin (cadherin-associated protein), delta 1. Cell-to-cell adhesion, signal transduction. 36,61. 15,96. '%(). Intercellular adhesion molecule 1. Typically expressed on endothelial and immune cells. 3,67A. -3,80A. NCAM1. Neural cell adhesion molecule 1. Cell-to-cell and cell-ECM interactions during cell differentiation.. -15,38A. 1,08A. *%(). Platelet/endothelial cell adhesion molecule. Leukocyte migration, angiogenesis, integrin activation. 74,23. 43,31. +*/*. Selectin E. Cell proliferation and migration. 34,15. 11,16. +*//. Selectin L. Mostly expressed on endothelial cells, recruitment of immune cells. 136,62. 122,22. +*/. Selectin P (granule membrane protein 140kDa, antigen CD62). Induction of hematopoiesis, expressed mostly on endothelial cells. 63,00. 13,99. +%*. Sarcoglycan, epsilon. Linking the actin cytoskeleton to the ECM; marker of differentiation in ESC. 1,67. 8,28. THBS1. Thrombospondin 1. 4,40. 26,54. THBS2. Thrombospondin 2. Cell-to-cell and cell-ECM interactions. 116,43A. Differentiation of hematoendothelial cells, organogenesis. -10,72. 1,67. Vascular cell adhesion molecule 1. Cell-cell adhesion adhesion, miogenesis. 240,07. 227,54. Versican. Proliferation, migration and angiogenesis, tissue morphogenesis. -1,28. 3,31. Thrombospondin 3. TNC. Tenascin C. VCAM1 VCAN. A. -6,65. 99,32. THBS3. Genes for integrins 3 (%

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(635) P R A C E O R Y G I N A L N E poł ożn i ct wo. DOI: 10.17772/gp/60977. Ginekol Pol. 2016, 87, 42-53. Marcin Tomsia et al. Gene expression of extracellular matrix components in human amniotic cells – a pilot study.. Table III. Expression of genes of ECM-associated proteins in amniotic cells in relation to differentiated cells. Genes CLEC3B and SPP1 are strongly up-regulated in amniotic cells (shaded boxes). A, This gene’s average threshold cycle is relatively high (> 30) in either the control or the test sample, and is reasonably low in the other sample (< 30). These data mean that the gene’s expression is relatively low in one sample and reasonably detected in the other sample. HA, hyaluronic acid.. . . . vs.

(636) . vs. keratinocytes. 679,03A. 3524,82A. %/*%:;. C-type lectin domain family 3, member B. Tissue remodeling. *%(). Extracellular matrix protein 1. Not fully elucidated role in skin differentiation, endochondral bone formation and angiogenesis. -1,97. 3,31. HAS1. Hyaluronan synthase 1. Synthesis of proangiogenic HA. 31,43. 11,74. </). Kallmann syndrome 1 sequence. Cell migration. 23,27. -3,41A. SPARC. Secreted protein, acidic, cysteine-rich (osteonectin). Counteradhesion and antiproliferation, antiangiogenic properties. 10,49. 31,78. +=. Spastic paraplegia 7 (pure and complicated autosomal recessive). Mitochondrial metalloprotease; marker of neurogenesis. 10,51. 6,70. SPP1. Secreted phosphoprotein 1. Marker of pluripotency, hyaluronan binding. 297,62. 107,39. &;'. Transforming growth factor, betainduced, 68kDa. Regulation of cell proliferation, differentiation and migration. -2,47. -1,11. Discussion 3 *

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