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Gene expression and canonical pathways in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells

Luciana Damous1, Juliana S. Nakamuta2, Ana E. Saturi De Carvalho2, Katia C. Carvalho1, Manuel J. Simões3, José M. Soares-Jr1, José E. Krieger2 and Edmund C. Baracat1
  • 1 Faculdade de Medicina da Universidade de São Paulo, Obstetrics and Gynecology, Brazil
  • 2 Instituto do coração, Faculdade de Medicina da Universidade de São Paulo, Laboratório de Cardiologia Genética e Molecular, Brazil
  • 3 Universidade Federal de São Paulo, Morphology, Brazil

Introduction: Previous works of our group demonstrated that cellular therapy with adipose tissue-derived stem cells (ASC) in a scaffold-based delivery is better than  the directly injection for improve the quality of frozen-thawed ovarian autografts[1],[2]. However, new researchers are necessary to evaluate the gene profile expressed in this graft to ensure that this therapy.

Materials and Methods: Twelve 12-week-old adult female Wistar rats were use. Frozen-thawed ovarian grafts of adult female rats were treated with rat ASC delivery in an acellular matrix (Gelfoam) immediately after an autologous retroperitoneal transplant (n=6). Controls received Gelfoam with vehicle (DMEM low glucose, n=6). The ovarian grafts were retrieved 30 days after transplantation. Quantitative gene expression (qPCR) for apoptosis, angiogenesis and inflammatory cytokines (84 genes in each pathway) were evaluated by Real Time-Polymerase Chain Reaction (RT-PCR). All reactions were performed in triplicate and controlled by negative RT (no enzyme) and no-template controls and all gene expression levels were normalized to the mean of the internal genes. A Fold Regulation reference value of -2 and +2 was considered for expression analyses. Further analyses were done by Ingenuity Pathway Analysis (IPAÒ) software in order to determine the main canoninal pathways and the expression values of the top molecules.

Results and Discussion: The top canonical pathways involved were Tweak Signaling (53%), Induction of Apoptosis (43%), Apoptosis Signaling (32%), Death Receptor Signaling (30%) and Hepatic fibrosis (17%). The top diseases and biofunctions envolved were: Organismal Injury and Abnormalities (121 molecules), Cancer (118), Inflammatory Response (108), Connective Tissue Disorders (76) and Tumor Morphology (75). The top tox functions were: Increased Levels of Alkaline Phosphatase (11 molecules), AST (7), ALT (6) and Creatinine (6) and Decreased Levels of Albumin (5). The 10 top molecules up-regulated, in descending order, were HPSE (+16.4 Fold Change), IL21 (+9.9), S1PR1 (+8.4), IL1A (+7.1), HGF (+7), CXCL11 (+6.7), CCL19 (+6.4), CCR3 (+6.2), CCR6 (+5.4) and LTB (+5.1). The 10 top molecules down-regulated, in descending order, were Hrk (-73.2), CASP14 (-71.1), TP73 (-34.1), DFFB (-27.8), DP63 (-26), CIDEB (-25.7), LTA (-23.6), BCL2L11 (-19.8), CD40LG (-18.5) and CIDEA (-17.8).

Conclusion: ASC therapy based on scaffold base-delivery strategy in rat ovarian autografts activated canonical pathways mainly involved with apoptosis. New researchers are necessary to evaluate the real benefit of this treatment.

São Paulo Research Foundation (Process numbers 2010/17897-5 and 2012/09469-9); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for post-doctoral scholarship

References:
[1] Damous LL, Nakamuta JS, Saturi de Carvalho AET, Carvalho KC, Soares-Jr JM, Simões MJ, Krieger JE, Baracat EC. Scaffold base-delivery of adipose tissue-derived stem cells in rat frozen-thawed ovarian autografts: preliminary studies in a rat model. J Assist Reprod Genet. 2015 Aug;32(8):1285-94. doi: 10.1007/s10815-015-0527-x. Epub 2015 Jul 24.
[2] Damous LL, Nakamuta JS, Saturi de Carvalho AET, Soares-Jr JM, Simões MJ, Krieger JE, Baracat EC. Adipose tissue-derived stem cells in rat cryopreserved ovarian grafts. Stem Cell Res There. 2015 6:57. DOI 10.1186/s13287-015-0068-3.

Keywords: stem cell, Biodegradable material, in vivo tissue engineering, acellullar matrix

Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.

Presentation Type: Poster

Topic: Synthetic scaffolds as extracellular matrices

Citation: Damous L, Nakamuta JS, Saturi De Carvalho AE, Carvalho KC, Simões MJ, Soares-Jr JM, Krieger JE and Baracat EC (2016). Gene expression and canonical pathways in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00399

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Received: 27 Mar 2016; Published Online: 30 Mar 2016.