Project description:Throught an reiterative growth factor and chemical screening, we defined a small molecule and growth factor cocktail, including EGF, glycogen synthase kinase 3 inhibitor (CHIR99021), transforming growth factor β receptor inhibitor (e.g. E-616452), lysophosphatidic acid and sphingosine 1-phosphate, that can sustain long-term self-renewal of murine hepatoblasts under chemically defined conditions. The expandable hepatoblasts (eHBs) by this small molecule and growth factor cocktail expressed a set of genes typical of liver progenitor cells and liver development, and retained the ability to respond to liver developmental cues and produce functional hepatocytes and form bile duct-like structures. Moreover, both early- and late-passage eHBs demonstrated a similar transcriptome profile. Microarray analysis also confirmed that the gene expression of cultured cells resembled hepatoblasts.
Project description:GDNF-regulated gene expression was studied in cultures of actively self-renewing spermatogonial stem cells established from 6 day old male mice. GDNF is the essential growth factor regulating mouse spermatogonial stem cell self-renewal. Using a serum-free chemically defined culture system that supports mouse spermatogonial stem cell self-renewal for extended periods of time, GDNF-regulated genes were identified using microarray profiling. Keywords: GDNF withdrawal and time-course replacement
Project description:GDNF-regulated gene expression was studied in cultures of actively self-renewing spermatogonial stem cells established from 6 day old male mice. GDNF is the essential growth factor regulating mouse spermatogonial stem cell self-renewal. Using a serum-free chemically defined culture system that supports mouse spermatogonial stem cell self-renewal for extended periods of time, GDNF-regulated genes were identified using microarray profiling. Experiment Overall Design: Established cultures of highly enriched self-renewing spermatogonial stem cells were subjeted to withrawal of GDNF and GFRalpha1 for 18-hr followed by replacement of the growth factors for 2, 4, and 8-hr. Gene expression was studied using microarray profiling prior to withdrawal, after withdrawal and at each time-point of GDNF/GFRalpha1 replacement.
Project description:Human induced hepatic lineage-oriented stem cells (hiHSCs) can be established as a new type of human induced pluripotent stem cells (hiPSCs) with only some modifications of their culture conditions in both generation and self-renewal. hiHSCs were generated and expanded at a very high density under non-typical coculture with feeder cells in a chemically defined hiPSC medium . In detail, hiHSCs were generated from human non-hepatic cells by gene transfer with pantropic retrovirus vectors carrying the three transcription factor genes OCT3/4, SOX2, and KLF4. They were expanded at a very high density under non-typical coculture with mouse embryonic fibroblasts (MEF) in a chemically defined mTeSR1 medium on gelatin-coated dishes. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. The potential hepatic specification of hiHSCs was defined by their expression profiles of hepatic markers under a self-renewal culture.
Project description:Comparison of H9 human embryonic stem cells, grown in chemically defined and animal-product free conditions, with their differentiated progeny - mesendodermal progenitors arisen as the result of changes made to chemically defined hESC growth conditions. Contributes to a larger work on the use of chemically defined growth conditions to direct hESC differentiation.
Project description:Here we present a strategy to adapt hESCs to high-throughput screening (HTS) conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several currently marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice. Global gene expression analysis upon drug treatment reveals overlapping and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the available repertoire of chemical compounds for manipulating hESC fate. Keywords: Global gene expression analysis, drug response, human ES cells, pluripotency, differentiation, self-renewal
Project description:Stat3+/+ and -/- ES cells were cultured in defined conditions and exposed to leukemia inhibitory factor (LIF) in a 24h timecourse to identify Stat3-independent targets of the self-renewal response to LIF signaling.
Project description:In this study we have analyzed the global gene expression of naïve mouse embryonic stem cells in different culture conditions including R2i (PD0325901+SB431542), 2i (PD0325901+CHIR99021), and also PD0325901+LIF and SB431542+LIF to show the similarities and differences between the conditions in maintaining pluripotency. Since the first generation of mouse embryonic stem (ES) cells, extrinsic regulation of pluripotency has been at the focus of attention. Here we show that suppression of transforming growth factor β (TGFβ) signaling could have impressive effects on self-renewal and pluripotency of mouse ES cells. We introduce a chemically defined medium with inhibitors of TGFβ and mitogen-activated protein kinase (MAPK) kinase, designated here as R2i (Royan 2 inhibitors), for highly efficient establishment of ES cell lines from various mouse strains. R2i also supports homogenous expression of Nanog and Dppa3 proteins in ES cells and shows minimal differentiation leakage. Our results uncover an appropriate condition for ES cell generation from single blastomeres of various cleavage-stage embryos. Further, the high accuracy of genome integrity after long-term cultivation in R2i illustrates an optimal condition for ES cell culture. Global transcriptomic analysis of R2i cells demonstrates that bone morphogenetic protein 4 (BMP4) signaling becomes overrepresented pursuant to TGFβ repression, which may confer further robustness to pluripotency through shielding cells from differentiation stimuli. These findings point to a new facet of ground state pluripotency by blocking differentiation pathways, without influencing the pluripotency regulatory circuitry of mouse ES cells. Whole gene expression study of 4 different mouse embryonic stem cell lines maintained under 4 different chemically defined conditions: R2i, 2i, PD and SB
Project description:Both FGF and WNT pathways play important roles in embryonic development, stem cell self-renewal and are frequently deregulated in breast cancer. To study the cooperation between FGF and WNT signaling, we have generated a mouse model, MMTV-WNT1/MMTV-iFGFR1 (WNT/iR1), in which we could chemically overactivate iFGFR1 in a ligand-independent manner.
Project description:Hematopoietic stem cells (HSCs) are identified by their ability to sustain prolonged blood cell production in vivo, although recent evidence suggests that durable self-renewal (DSR) is shared by HSC subtypes with distinct self-perpetuating differentiation programs. Net expansions of DSR-HSCs occur in vivo, but molecularly defined conditions that support similar responses in vitro are lacking. We hypothesized that this might require a combination of factors that differentially promote HSC viability, proliferation and self-renewal. We now demonstrate that HSC survival and maintenance of DSR potential is variably supported by different Steel factor (SF)-containing cocktails with similar HSC-mitogenic activities. In addition, stromal cells produce other factors, including nerve growth factor and collagen 1, that can antagonize the apoptosis of initially quiescent adult HSCs and, in combination with SF and interleukin-11, produce >15-fold net expansions of DSR-HSCs ex vivo within 7 days. These findings suggest a new molecular basis for HSC control and expansion. Adult mouse bone marrow CD45+EPCR+CD48-CD150+ cells were used in a total of 4 conditions (fresh, 6 hour stimulation with SF+IL-11+UG26 CM, UG26 CM only, SF+IL-11) with 2 technical replicates per condition.