Project description:Proper regulation of the proliferation and differentiation of radial glia (RG), the neural stem cells of the developing cortex, is fundamental for brain growth and organization. In humans, a specialized RG subtype, the outer radial glia (oRG), are abundant and give rise to diverse neuronal and glial progeny. However, the mechanisms regulating oRG development and differentiation are not fully understood. Here we investigated the regulation of oRG expansion and lineage potential by perturbing Leukemia Inhibitory Factor (LIF) signaling in the developing human cortex and in human pluripotent stem cell (PSC)-derived cortical organoids. LIF receptors are specifically expressed in oRG cells during neurogenesis, and, consistent with the previously described role of this cytokine as an activator of stem cell self-renewal, LIF treatment increased the number of oRG cells. Surprisingly, LIF treatment also increased the production of inhibitory interneurons (INs) in the cortex. Comparative transcriptomic analysis suggested that these INs resemble INs produced in the caudal ganglionic eminence (CGE). To test if oRG cells are the progenitors of these CGE-like INs, we isolated oRG cells from primary developing cortex and cultured them for several weeks with and without LIF treatment. We found that CGE-like INs were produced by oRG cells, and their abundance is increased by LIF treatment. Together, these observations suggest that LIF signaling regulates oRG lineage potential and capacity to generate CGE-like INs.
Project description:We report that ES cells cultured in ground state (2i and 2i/LIF) culture conditions are heterogeneous and show heterogeneus expression of extraembryonic markers. Using a highly sensitive reporter for the endoderm marker Hex we can sort Hex high and low populations from either serum/LIF or 2i/LIF and demonstrate that they have different functional properties. Here we explored the transcriptional basis of these functional differences and noted that Hex low (HV-) and Hex high (HV+) populations showed more distinct expression profiles in 2i/LIF than in serum/LIF. Additionally in 2i/LIF the HV+ population showed an upregulation of extraembryonic markers (such as trophoblast stem cell specific genes) and also imprinted genes compared to the HV- population, which is not observed when these populations are sorted from serum/LIF. We also analysed the transcriptional effect of LIF in 2i by analysing unsorted ES cells cultured in either 2i alone or 2i with LIF. We observed that the addition of LIF led to an upregulation of extraembryonic markers but did not effect the expression of pluripotency genes, other than Klf4. Additionally, the most significantly upregulated genes from 2i/LIF cultured ES cells compared to 2i cultured ES cells showed the greatest correlation to placental tissue when compared to the GNF tissue specific expression database. This analysis, alongside functional experiments, suggested that HV+ ES cells in 2i/LIF corresponded to an extraembryonically primed population of cells and that the addition of LIF supported this population.
Project description:LIF has an important role in immunosupression in different scenarios, such as in embryo implantation in the uterus, autoimmune disease or organ transplantation. In tumor progression it has been largely demonstrated the importance of immune system. The fact that LIF is highly expressed in certain tumor types, in addition to its immunomodulatory properties, led us to hypothesize that tumors expressing high levels of LIF might be promoting an immune-tolerant microenvironment precluding the anti-tumor immune response. To establish whether the effect of LIF was relevant in the context of human monocytes, we isolated CD14+ cells (monocytes) from peripheral blood mononuclear cells (PBMCs) from healthy donors and analyzed the effect of LIF blockade. Instead of using recombinant LIF, we studied the effect of the endogenous LIF secreted by U251 cell line. RNA was extracted from the CD14+ cells and the transcriptomic analysis was performed.
Project description:Lenalidomide, an IMiD® immunomodulatory agent used for the treatment of multiple myeloma (MM),is believed to target the stromal support, but its precise mechanism on the phenotype or the effector functions of macrophages is still unclear. To investigate the effect of lenalidomide on macrophages, M-CSF generated macrophages were treated with Lenalidomide and analyzed by RNA-seq.