Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-m porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal versus disease-related endometrial microenvironments

Analysis of the role of the transcription factor C/EBPβ in controlling uterine functions during early pregnancy

Embryo implantation into the endometrium is a complex biological process involving the integration of steroid hormone signaling, endometrial tissue remodeling and maternal- fetal communications. A successful pregnancy is the outcome of the timely integration of these events during the early stages of implantation. The involvement of ovarian steroid hormones, estrogen (E) and progesterone (P), acting through their cognate receptors, is essential for uterine functions during pregnancy. The molecular mechanisms that control the process of implantation are undergoing active exploration. Through our recent efforts, we identified the transcription factor, CCAAT Enhancer Binding Protein Beta (C/EBPb) as a prominent target of estrogen and progesterone signaling in the uterus. The development of a C/EBPb-null mouse model, which is infertile, presented us with an opportunity to analyze the role of this molecule in uterine function. We discovered that C/EBPb functions in two distinct manners: (i) by acting as a mediator of E-induced proliferation of the uterine epithelium and (ii) by controlling uterine stromal cell differentiation, a process known as decidualization, during pregnancy. My studies have delineated important mechanisms by which E regulates C/EBPb expression to induce DNA replication and prevent apoptosis of uterine epithelial cells during E-induced epithelial growth. In subsequent studies, I analyzed the role of C/EBPb in decidualization and uncovered a unique mechanism by which C/EBPb regulates the synthesis of a unique laminin-containing extracellular matrix (ECM) that supports stromal cell differentiation and embryo invasion. In order to better define the role of laminin in implantation, we developed a laminin gamma 1-conditional knockout mouse model. This is currently an area of ongoing investigation. The information gained from our analysis of C/EBPb function in the uterus provides new insights into the mechanisms of steroid hormone action during early pregnancy. Ultimately, our findings may aid in the understanding of dysregulation of hormone-controlled pathways that underlie early pregnancy loss and infertility in women

Analysis of the Role of the Transcription Factor C/EBPbeta in Controlling Uterine Functions During Early Pregnancy

61 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2010.Embryo implantation into the endometrium is a complex biological process involving the integration of steroid hormone signaling, endometrial tissue remodeling and maternal-fetal communications. A successful pregnancy is the outcome of the timely integration of these events during the early stages of implantation. The involvement of ovarian steroid hormones, estrogen (E) and progesterone (P), acting through their cognate receptors, is essential for uterine functions during pregnancy. The molecular mechanisms that control the process of implantation are undergoing active exploration. Through our recent efforts, we identified the transcription factor, CCAAT Enhancer Binding Protein Beta (C/EBPbeta) as a prominent target of estrogen and progesterone signaling in the uterus. The development of a C/EBPbeta-null mouse model, which is infertile, presented us with an opportunity to analyze the role of this molecule in uterine function. We discovered that C/EBPbeta functions in two distinct manners: (i) by acting as a mediator of E-induced proliferation of the uterine epithelium and (ii) by controlling uterine stromal cell differentiation, a process known as decidualization, during pregnancy. My studies have delineated important mechanisms by which E regulates C/EBPbeta expression to induce DNA replication and prevent apoptosis of uterine epithelial cells during E-induced epithelial growth. In subsequent studies, I analyzed the role of C/EBPbeta in decidualization and uncovered a unique mechanism by which C/EBPbeta regulates the synthesis of a unique laminin-containing extracellular matrix (ECM) that supports stromal cell differentiation and embryo invasion. In order to better define the role of laminin in implantation, we developed a laminin gamma1-conditional knockout mouse model. This is currently an area of ongoing investigation. The information gained from our analysis of C/EBPbeta function in the uterus provides new insights into the mechanisms of steroid hormone action during early pregnancy. Ultimately, our findings may aid in the understanding of dysregulation of hormone-controlled pathways that underlie early pregnancy loss and infertility in women.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes

Differentially expressed genes within 1 Mb of <i>FOSB</i>.

Differentially expressed genes within 1 Mb of FOSB.</p

Expression of genes within 1 Mb of rs148726219.

Expression values are expressed as fragments per kilobase per million mapped reads (FPKM) for WT-2A1 (green), rs148726219-heterozygous (HET-2D2, HET-2G6; orange), and homozygous (HOM-2B11, HOM-2H6; purple) clones at day 0, 2, 6, 13, and 23 of hiPSC-iNeuron differentiation. Four technical replicates for each clone are shown with each represented by a black dot. A. ERCC1. B. BCL3. C. PPP1R37. D. RTN2. E. DMPK. F. APOE. (PDF)</p

Additional western blots for catalase expression used to perform quantification.

Additional western blots of day 23 iNeuron lysates from independent hiPSC-iN differentiations of BIONi010-C-13 parental, WT-2A1, HET-2D2, HET-2G6, HOM-2B11, and HOM-2H6 clones. Blots were probed with an antibody against catalase and GAPDH as a loading control. The first experimental replicate is shown in Fig 6. A. Second experimental replicate. B. Third experimental replicate. (PDF)</p

Gene expression measured by SYBR Green RT-qPCR.

Values for BIONi010-C-13 WT-parental line and WT-2A1 clone (grey bars), rs148726219-heterozygous clones (blue bars), and homozygous clones (green bars) day 0, 2, 6, 13, and 23 of hiPSC-iNeuron differentiation are shown. Cq values were normalized to the geometric mean of 3 housekeeping genes (B2M, ACTB, GAPDH) and where possible, are expressed relative to the WT-parental line. A. Total FOSB transcript. Day 0, n = 3; Day 2, n = 2; Day 23, n = 4. B. DFOSB. Day 0, n = 3; Day 2, n = 2; Day 23, n = 4. C. Total ERCC1 transcript. Day 0, n = 3; Day 2, n = 2; Day 23, n = 4. D. ERCC1 long transcript isoform. Day 0, n = 3; Day 2, n = 2; Day 23, n = 4. E. CAT. n = 2. F. PCDHB5. n = 2. (PDF)</p