Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme.

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

Identifying Biomarkers of Wharton's Jelly Mesenchymal Stromal Cells Using a Dynamic Metabolic Model: The Cell Passage Effect.

Because of their unique ability to modulate the immune system, mesenchymal stromal cells (MSCs) are widely studied to develop cell therapies for detrimental immune and inflammatory disorders. However, controlling the final cell phenotype and determining immunosuppressive function following cell amplification in vitro often requires prolonged cell culture assays, all of which contribute to major bottlenecks, limiting the clinical emergence of cell therapies. For instance, the multipotent Wharton's Jelly mesenchymal stem/stromal cells (WJMSC), extracted from human umbilical cord, exhibit immunosuppressive traits under pro-inflammatory conditions, in the presence of interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα). However, WJMSCs require co-culture bioassays with immune cells, which can take days, to confirm their immunomodulatory function. Therefore, the establishment of robust cell therapies would benefit from fast and reliable characterization assays. To this end, we have explored the metabolic behaviour of WJMSCs in in vitro culture, to identify biomarkers that are specific to the cell passage effect and the loss of their immunosuppressive phenotype. We clearly show distinct metabolic behaviours comparing WJMSCs at the fourth (P4) and the late ninth (P9) passages, although both P4 and P9 cells do not exhibit significant differences in their low immunosuppressive capacity. Metabolomics data were analysed using an in silico modelling platform specifically adapted to WJMSCs. Of interest, P4 cells exhibit a glycolytic metabolism compared to late passage (P9) cells, which show a phosphorylation oxidative metabolism, while P4 cells show a doubling time of 29 h representing almost half of that for P9 cells (46 h). We also clearly show that fourth passage WJMSCs still express known immunosuppressive biomarkers, although, this behaviour shows overlapping with a senescence phenotype.

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.