Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of Transcription Factor Networks Involved in Umbilical Cord Blood CD34+ Stem Cells-Derived Erythropoiesis

ABSTRACT: The characteristics of global gene expression patterns during umbilical cord blood (UCB)-CD34+ stem cell-derived erythropoiesis are not clearly elucidated. In this study, UCB-stem cells were grown in liquid culture as a model for this process. We observed a high proliferative capacity of UCB-stem cells producing over 10 billion viable cells in culture. Normal erythroid maturation was confirmed by increased expression of CD71 and CD235a biomarkers. Furthermore, the gamma- to beta-globin gene switch was observed around day 45 indicating extended gamma-globin gene expression in fetal erythroid cells. To study global gene expression patterns, microarray analysis was performed on days 21, 42, 49 and 56. Forty-five transcription factors were silenced during the culture period (Profile-1) including CUX1 and HES5 among others. Conversely, 30 transcription factors were activated by day 56 (Profile-2) such as KLF1, GATA1, and MAFB. Both datasets were analyzed further using the MIMI Cytoscape platform which defined transcription factor networks around KLF4 and GATA2 from Profile-1 genes and KLF1 and GATA1 for Profile-2 genes. The characteristics of UCB-CD34+ stem cells including high proliferative capacity and prolonged gamma-globin expression combined with novel transcription factor networks suggest novel mechanisms of fetal UCB-stem cell-derived erythropoiesis. Cd34 cell from umbilical cord blood were grown in three independent cultures using the one-phase protocol. Cells were cultured in aMEM containing 30% fetal bovine serum, 1% deionized BSA with penicillin (100 U/mL) and streptomycin (0.1 mg/mL) at 37°C and 5% CO2. The following growth factors were added on day 0: stem cell factor (50 ng/mL), interleukin-3 (10 ng/mL) and erythropoietin (4 U/mL). Approximately 1.5 million cells were harvested from each culture (triplicate samples) on day 21, 42, 49 and 56 for microarray analysis on the whole-genome Illumina HumanWG-12 V4 Expression BeadChip.

ORGANISM(S): Homo sapiens

SUBMITTER: Biaoru Li

PROVIDER: E-GEOD-49438 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Introduction Fetal microchimerism could be involved in the regulation of breast cancer oncogenesis. CD34+ cells could be of a particular interest as up to 12% of the CD34+ population in maternal blood are of fetal origin. The aim of this research was to analyze the impact of umbilical cord blood (UCB) CD34+ on MCF-7 and MDA-MB-231 breast cancer cell lines. Material and Methods UCB CD34+ cells were obtained from healthy women at full-term delivery. Direct and indirect cultures were grown with MCF-7 and MDA-MB-231 cells. Proliferation, migration, invasion, and transcriptomic analysis of breast cancer cell lines were compared between cultures exposed and non-exposed to UCB CD34+ cells. Interactions between UCB CD34+ and breast cancer cells were analyzed under fluorescent microscopy. Functional analyses were generated with QIAGEN’s Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Results Direct contact between UCB CD34+ and breast cancer cell lines induced a reduction in the proliferative capacities of MCF-7 and MDA-MB-231 and diminished the migration abilities of MDA-MB-231 cells. In 3D co-culture, UCB CD34+ cells were attracted by tumor spheroids and incorporated into tumor cells. These cell-to-cell interactions were responsible for transcriptome modifications coherent with observed functional modifications. Among the cytokines secreted by UCB CD34+, INF- was identified as a potential upstream regulator responsible for the molecular modifications observed in transcriptomic analysis of MCF-7 breast cancer cells exposed to UCB CD34+ cells, as was IL-17A in MDA-MB-231 cells. Conclusion Direct cell-to-cell contact induces functional modifications in breast cancer cells. Interactions between UCB CD34+ and breast cancer cells could induce cell fusion and signal transmission via cytokines. Further analysis of direct cell-to-cell interactions should be performed at a molecular level to further understand the potential role of fetal CD34+ cells in breast cancer.

Project description:The fetal and adult globin genes in the human b-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal g-globin (HBG) to replace abnormal adult sickle bS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of g-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize protein expression patterns during the g-globin to b-globin (g/b) switch observed throughout in vitro erythroid differentiation. We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the g/b-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal g-globin expression at day 7 with a switch to a predominance of b-globin expression by day 28 and the g/b-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated >1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kb, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in g-and b-globin regulation were identified. The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is a good model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation. Peripheral blood mononuclear cells were isolated from three normal donors using Histapaque-1.077. The mononuclear cells were grown in three independent cultures using the one-phase protocol as previously published. Cells were cultured in alphaMEM containing 30% fetal bovine serum, 1% deionized BSA with penicillin (100 U/mL) and streptomycin (0.1 mg/mL) at 37°C and 5% CO2. The following growth factors were added on day 0: stem cell factor (50 ng/mL), interleukin-3 (10 ng/mL) and erythropoietin (4 U/mL). Approximately 1.5 million cells were harvested from each culture (triplicate samples) on day 7, 14, 21 and 28 for microarray analysis on the whole-genome Illumina HumanWG-6 V2 Expression BeadChip.

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Characterization of Transcription Factor Networks Involved in Umbilical Cord Blood CD34+ Stem Cells-Derived Erythropoiesis

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