CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies.

Background: Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations - demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency - enabling unlimited production of modified "off-the-shelf" hPSC-NKs. Methods: hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro. Results: HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions: Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research - improving patient survival and quality of life.

iPSC-derived progenitor stromal cells provide new insights into aberrant musculoskeletal development and resistance to cancer in down syndrome.

Down syndrome (DS) is a congenital disorder caused by trisomy 21 (T21). It is associated with cognitive impairment, muscle hypotonia, heart defects, and other clinical anomalies. At the same time, individuals with Down syndrome have lower prevalence of solid tumor formation. To gain new insights into aberrant DS development during early stages of mesoderm formation and its possible connection to lower solid tumor prevalence, we developed the first model of two types of DS iPSC-derived stromal cells. Utilizing bioinformatic and functional analyses, we identified over 100 genes with coordinated expression among mesodermal and endothelial cell types. The most significantly down-regulated processes in DS mesodermal progenitors were associated with decreased stromal progenitor performance related to connective tissue organization as well as muscle development and functionality. The differentially expressed genes included cytoskeleton-related genes (actin and myosin), ECM genes (Collagens, Galectin-1, Fibronectin, Heparan Sulfate, LOX, FAK1), cell cycle genes (USP16, S1P complexes), and DNA damage repair genes. For DS endothelial cells, our analysis revealed most down-regulated genes associated with cellular response to external stimuli, cell migration, and immune response (inflammation-based). Together with functional assays, these results suggest an impairment in mesodermal development capacity during early stages, which likely translates into connective tissue impairment in DS patients. We further determined that, despite differences in functional processes and characteristics, a significant number of differentially regulated genes involved in tumorigenesis were expressed in a highly coordinated manner across endothelial and mesodermal cells. These findings strongly suggest that microRNAs (miR-24-4, miR-21), cytoskeleton remodeling, response to stimuli, and inflammation can impact resistance to tumorigenesis in DS patients. Furthermore, we also show that endothelial cell functionality is impaired, and when combined with angiogenic inhibition, it can provide another mechanism for decreased solid tumor development. We propose that the same processes, which specify the basis of connective tissue impairment observed in DS patients, potentially impart a resistance to cancer by hindering tumor progression and metastasis. We further establish that cancer-related genes on Chromosome 21 are up-regulated, while genome-wide cancer-related genes are down-regulated. These results suggest that trisomy 21 induces a modified regulation and compensation of many biochemical pathways across the genome. Such downstream interactions may contribute toward promoting tumor resistant mechanisms.

Down syndrome iPSC model: endothelial perspective on tumor development.

Trisomy 21 (T21), known as Down syndrome (DS), is a widely studied chromosomal abnormality. Previous studies have shown that DS individuals have a unique cancer profile. While exhibiting low solid tumor prevalence, DS patients are at risk for hematologic cancers, such as acute megakaryocytic leukemia and acute lymphoblastic leukemia. We speculated that endothelial cells are active players in this clinical background. To this end, we hypothesized that impaired DS endothelial development and functionality, impacted by genome-wide T21 alterations, potentially results in a suboptimal endothelial microenvironment with the capability to prevent solid tumor growth. To test this hypothesis, we assessed molecular and phenotypic differences of endothelial cells differentiated from Down syndrome and euploid iPS cells. Microarray, RNA-Seq, and bioinformatic analyses revealed that most significantly expressed genes belong to angiogenic, cytoskeletal rearrangement, extracellular matrix remodeling, and inflammatory pathways. Interestingly, the majority of these genes are not located on Chromosome 21. To substantiate these findings, we carried out functional assays. The obtained phenotypic results correlated with the molecular data and showed that Down syndrome endothelial cells exhibit decreased proliferation, reduced migration, and a weak TNF-α inflammatory response. Based on this data, we provide a set of genes potentially associated with Down syndrome's elevated leukemic incidence and its unfavorable solid tumor microenvironment-highlighting the potential use of these genes as therapeutic targets in translational cancer research.

Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells.

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation.