Human primordial germ cells (hPGCs) emerge as a precursor cell type for mature gametes in the reproductive system during embryogenesis. The formation of human primordial germ cell-like cells (hPGCLCs) via in vitro embryo models gives researchers the opportunity to study hPGC development in a non-invasive manner. Here, we describe the optimization of a peri-gastruloid model to study hPGCLC formation in an environment that recapitulates spatiotemporal aspects of hPGCLC induction. This embryo model mimics the post-implantation stages of development in vivo through early organogenesis, which includes hPGCLC formation. Two different embryonic stem cell (hESC) lines, RUES2-GLR and STAG 18, were used to analyze the formation of embryonic structures and hPGCLCs, respectively. Morphological analysis from the experimental data shows the formation of hypoblast-like and epiblast-like structures in the models. Immunofluorescence staining (IF) indicates the presence of hPGCLCs that are triple positive for the hPGCLC genetic markers BLIMP1, TFAP2C, and SOX17. In addition, qPCR analysis of hPGCLCs display a relative increase in genetic expression of early hPGCLC markers TFAP2C, SOX17, POU5F1, NANOG, and NANOS3. Despite cell line specific variation in peri-gastruloid generation, hPGCLCs successfully develop within the embryo model and can be harvested for further genetic analysis. Research on precursory germ cells expands current understandings of infertility and promotes the expansion of family planning options for all individuals.

Breast cancer (BC) is the most common cancer worldwide and disproportionately affects African American women (AAW) compared to European American women (EAW). AAW face earlier onset, more aggressive subtypes, and lower survival rates. Genetic analyses have shown BC tumors from AAW show distinct genetic profiles and immune signatures suggesting an immunosuppressive tumor microenvironment (TME). It has been described BC induces aberrant myelopoiesis and accumulation of myeloid-derived suppressor cells (MDSCs) that contribute to immune evasion by tumors. We previously reported that estrogen signaling expands MDSCs, an effect blocked by selective estrogen receptor degraders (SERDs) like fulvestrant and our newly designed SERD JD128. In vitro luminex assay results showed BC cells from AAW secreted higher basal levels of proinflammatory cytokines, VEGF, and G-CSF compared to cells from EAW (P<0.05). Estradiol further increased secretion of proinflammatory cytokines, G-CSF, and PDGF-AA, stimulated proliferation of CD34+ hematopoietic stem cells and MDSCs, effects that were blocked by SERDs fulvestrant and JD128. Our results showed a significant reduction of these pro-tumor effects may be mediated through estrogen receptor alpha downregulation after SERDs treatment. Our findings suggest an estrogen-driven mechanism of immunosuppression. SERD-based therapies in combination with targeted therapies may benefit receptor positive and negative BC tumors.

Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) hold significant potential for applications in disease modeling, drug discovery, and regenerative medicine. However, differentiation efficiency and yield vary widely across protocols and cell lines, limiting the reproducibility and scalability of iPSC-derived cardiomyocytes. This project aimed to optimize a robust, chemically defined, albumin-free differentiation protocol specifically for iPSC lines obtained from the UCLA Broad Stem Cell Research Center. Three differentiation protocols were evaluated, each using stage-specific Wnt pathway modulation with varying media conditions and purification strategies. Timeline 1 uses a standard differentiation strategy using sequential Wnt activation and inhibition in CDM3 media. Timeline 2 incorporated Wnt inhibition with IWP2 and metabolic selection in RPMI+B27 media, improving purity but yielding less than 10% contracting area. Timeline 3 implemented an optimized differentiation strategy incorporating heparin, a novel Wnt signaling modulator, in a fully chemically defined system, followed by lactate-based metabolic selection. This protocol produced cardiomyocyte yields exceeding 50% contracting area. These results demonstrate that the combination of heparin-mediated Wnt modulation and metabolic selection significantly improves cardiomyocyte differentiation yield and reproducibility. This work provides an optimized platform for generating high-purity cardiomyocytes from iPSCs to support future applications.

The ZFP36 proteins (ZFP36, ZFP36L1, ZFP36L2) bind to AU-rich regions in the 3’untranslated region (UTR) of mRNA. They recruit deadenylation complexes to promote RNA degradation. The hepatokine Fgf21, a regulator of systemic metabolism, has three canonical regions, AUUUA, where ZFP36 proteins bind to post-transcriptionally regulate mRNA stability. We hypothesize that single nucleotide polymorphisms (SNP) within the 3’UTR would increase the stability of Fgf21 mRNA by disrupting ZFP36 protein binding, thus, reducing mRNA degradation. To test the hypothesis, we cloned the Fgf21 3’UTR downstream of a luciferase reporter. We included a wildtype (WT) 3’UTR, a delta mutant lacking all 3 ZFP36 protein binding sites, and SNPs identified in the human population. Then, we co-transfected the 3’UTR luciferase plasmids and increasing concentrations of ZFP36 proteins into HEK293 cells. We used a luciferase assay to quantify the mRNA stability of Fgf21 with increasing ZFP36 protein concentrations. The WT Fgf21 3’UTR showed a dose-responsive decrease in mRNA stability with increasing ZFP36 concentrations. In contrast, the delta mutant Fgf21 3’UTR was not responsive to the ZFP36 protein dose response and displayed increased stability. The SNP constructs displayed elevated luciferase activity, though lower than delta mutants, likely because a single binding site was disrupted. SNPs within the 3’UTR hinder the Fgf21 post-transcriptional regulation by interfering with ZFP36 protein binding–a key regulatory mechanism of Fgf21 expression.

Autophagy, a cellular degradation process, is essential for maintaining cellular homeostasis and responding to stress. Autophagy plays a complex role in cancer biology as it can suppress tumor initiation and yet support tumor growth in established cancers. Previous research found that whole-body loss of autophagy led to smaller tumors. However, the role of autophagy in specific cell populations within the tumor microenvironment (TME) remains elusive. Myeloid-derived cells such as macrophages and dendritic cells (DCs) play intricate roles within the TME and are capable of either promoting or inhibiting tumor progression, partially through their respective roles in T cell activation. We disrupted the autophagy pathway in macrophages and DCs to identify the function of autophagy in these cells during a tumor response. Macrophages and DCs were differentiated in vitro from bone marrow isolated from autophagy-proficient or deficient mice. The cultured cells were then treated with melanoma-conditioned media to simulate a TME, and the expression of proinflammatory cytokines was analyzed by RT qPCR. Autophagy-deficient macrophages and DCs expressed higher levels of proinflammatory cytokines than their autophagy-proficient counterparts. This heightened activity from autophagy-deficient macrophages and DCs could be essential in slowing cancer progression. Ultimately, these findings represent an important step towards a better understanding of the role of autophagy in cancer and potentially towards the development of new therapeutics.