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Development of a novel protocol for isolation and purification of human granulosa cells.

ABSTRACT: To develop an optimal method of isolation and purification of human granulosa cells from ovarian follicular fluid.Follicular fluid was collected from patients undergoing oocyte retrieval. A series of isolation and purification techniques was performed, involving density gradient centrifugation and use of different antibody-bead complexes.The highest percent yield of live purified granulosa cells came from density gradient centrifugation using sucrose polymer followed by positive selection of granulosa cells using primary antibody to MISRII and secondary antibody coupled to iron oxide beads.A novel protocol for granulosa cell purification has been developed yielding samples that are largely free of nondesirable cells. This protocol provides a purification solution, especially for patient samples that have significant RBC contamination.

SUBMITTER: Chilvers RA

PROVIDER: S-EPMC3370046 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Development of a novel protocol for isolation and purification of human granulosa cells.

Chilvers R A RA Bodenburg Y H YH Denner L A LA Urban R J RJ

Journal of assisted reproduction and genetics 20120310 6

<h4>Purpose</h4>To develop an optimal method of isolation and purification of human granulosa cells from ovarian follicular fluid.<h4>Methods</h4>Follicular fluid was collected from patients undergoing oocyte retrieval. A series of isolation and purification techniques was performed, involving density gradient centrifugation and use of different antibody-bead complexes.<h4>Results</h4>The highest percent yield of live purified granulosa cells came from density gradient centrifugation using sucro ...[more]

PMID: 22407611

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Project description:The forkhead box (FOX), FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown here, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation. Immunostaining and Western blot analyses confirmed FOXO1 and phosphatase and tensin homolog (PTEN) depletion, maintenance of globin transcription factor (GATA) 4 and nuclear localization of FOXL2 and phosphorylated small mothers against decapentaplegic (SMAD) 2/3 in the tumor cells, recapitulating results we observed in human adult GCTs. Microarray and quantitative PCR analyses of mouse GCTs further confirmed expression of specific genes (Foxl2, Gata4, and Wnt4) controlling granulosa cell fate specification and proliferation, whereas others (Emx2, Nr0b1, Rspo1, and Wt1) were suppressed. Key genes (Amh, Bmp2, and Fshr) controlling follicle growth, apoptosis, and differentiation were also suppressed. Inhbb and Grem1 were selectively elevated, whereas reduction of Inha provided additional evidence that activin signaling and small mothers against decapentaplegic (SMAD) 2/3 phosphorylation impact GCT formation. Unexpectedly, markers of Sertoli/epithelial cells (SRY [sex determining region Y]-box 9/keratin 8) and alternatively activated macrophages (chitinase 3-like 3) were elevated in discrete subpopulations within the mouse GCTs, indicating that Foxo1/3/Pten depletion not only leads to GCTs but also to altered granulosa cell fate decisions and immune responses. Thus, analyses of the Foxo1/3/Pten mouse GCTs and human adult GCTs provide strong evidence that impaired functions of the FOXO1/3/PTEN pathways lead to dramatic changes in the molecular program within granulosa cells, chronic activin signaling in the presence of FOXL2 and GATA4, and tumor formation.

Project description:BackgroundHigh levels of dopamine (DA) were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs) derived from women undergoing in vitro fertilization (IVF) are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality.MethodsCells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4) were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system.ResultsWe found members of the two DA receptor families (D1- and D2 -like) associated with different signaling pathways in human GCs, namely D1 (as expected) and D5 (both are Gs coupled and linked to cAMP increase) and D2, D4 (Gi/Gq coupled and linked to IP3/DAG). D3 was not found. The presence of the trophic hormone hCG (10 IU/ml) in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR) or protein levels (immunocytochemistry/Western blotting) of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S). Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed in the absence of extracellular calcium and was abolished by a D2 blocker (L-741,626). DA treatment (48 h) of human GCs resulted in slightly, but significantly enlarged, viable cells.ConclusionA previous study showed D2 in human GCs, which are linked to cAMP, and the present study reveals the full spectrum of DA receptors present in these endocrine cells, which also includes D2-like receptors, linked to calcium. Ovarian DA can act thus via D1,2,4,5, which are co-expressed by endocrine cells of the follicle and the corpus luteum and are linked to different signaling pathways. This suggests a complex role of DA in the regulation of ovarian processes.

Project description:Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, Fc?RI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

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Development of a novel protocol for isolation and purification of human granulosa cells.

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Development of a novel protocol for isolation and purification of human granulosa cells.

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