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.

The utility of stem cells in pediatric urinary bladder regeneration.

Pediatric patients with a neurogenic urinary bladder, caused by developmental abnormalities including spina bifida, exhibit chronic urological problems. Surgical management in the form of enterocystoplasty is used to enlarge the bladder, but is associated with significant clinical complications. Thus, alternative methods to enterocystoplasty have been explored through the incorporation of stem cells with tissue engineering strategies. Within the context of this review, we will examine the use of bone marrow stem cells and induced pluripotent stem cells (iPSCs), as they relate to bladder regeneration at the anatomic and molecular levels. The use of bone marrow stem cells has demonstrated significant advances in bladder tissue regeneration as multiple aspects of bladder tissue have been recapitulated including the urothelium, bladder smooth muscle, vasculature, and peripheral nerves. iPSCs, on the other hand, have been well characterized and used in multiple tissue-regenerative settings, yet iPSC research is still in its infancy with regards to bladder tissue regeneration with recent studies describing the differentiation of iPSCs to the bladder urothelium. Finally, we examine the role of the Sonic Hedgehog signaling cascade that mediates the proliferative response during regeneration between bladder smooth muscle and urothelium. Taken together, this review provides a current, comprehensive perspective on bladder regeneration.

Mapping mouse hemangioblast maturation from headfold stages.

The mouse posterior primitive streak at neural plate/headfold stages (NP/HF, ~7.5 dpc-8 dpc) represents an optimal window from which hemangioblasts can be isolated. We performed immunohistochemistry on this domain using established monoclonal antibodies for proteins that affect blood and endothelial fates. We demonstrate that HoxB4 and GATA1 are the first set of markers that segregate independently to endothelial or blood populations during NP/HF stages of mouse embryonic development. In a subset of cells, both proteins are co-expressed and immunoreactivities appear mutually excluded within nuclear spaces. We searched for this particular state at later sites of hematopoietic stem cell emergence, viz., the aorta-gonad-mesonephros (AGM) and the fetal liver at 10.5-11.5 dpc, and found that only a rare number of cells displayed this character. Based on this spatial-temporal argument, we propose that the earliest blood progenitors emerge either directly from the epiblast or through segregation within the allantoic core domain (ACD) through reduction of cell adhesion and pSmad1/5 nuclear signaling, followed by a stochastic decision toward a blood or endothelial fate that involves GATA1 and HoxB4, respectively. A third form in which binding distributions are balanced may represent a common condition shared by hemangioblasts and HSCs. We developed a heuristic model of hemangioblast maturation, in part, to be explicit about our assumptions.

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.

Lysine Deprivation during Maternal Consumption of Low-Protein Diets Could Adversely Affect Early Embryo Development and Health in Adulthood.

The conversion of lysine to glutamate is needed for signaling in all plants and animals. In mouse embryonic stem (mES) cells, and probably their progenitors, endogenous glutamate production and signaling help maintain cellular pluripotency and proliferation, although the source of glutamate is yet to be determined. If the source of glutamate is lysine, then lysine deprivation caused by maternal low-protein diets could alter early embryo development and, consequently, the health of the offspring in adulthood. For these reasons, we measured three pertinent variables in human embryonic stem (hES) cells as a model for the inner cell masses of human blastocysts. We found that RNA encoding the alpha-aminoadipic semialdehyde synthase enzyme, which regulates glutamate production from lysine, was highly expressed in hES cells. Moreover, the mean amount of lysine consumed by hES cells was 50% greater than the mean amount of glutamate they produced, indicating that lysine is likely converted to glutamate in these cells. Finally, hES cells expressed RNA encoding at least two glutamate receptors. Since this may also be the case for hES progenitor cells in blastocysts, further studies are warranted to verify the presence of this signaling process in hES cells and to determine whether lysine deprivation alters early mammalian embryo development.

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.

GLI1 genotypes do not predict basal cell carcinoma risk: a case control study.

BACKGROUND: Susceptibility to basal cell carcinoma results from complex interactions between ultraviolet radiation exposure and genetic factors. The GLI1 oncogene is believed to play a role in the genesis of these tumors. We determined whether GLI1 polymorphisms were risk factors for developing basal cell carcinoma, either alone or in combination with patterns of past sun exposure, and whether there were functional differences among different GLI1 haplotypes. RESULTS: GLI1 genotypes at c.2798 and c.3298 from 201 basal cell carcinoma patients were compared to 201 age and sex-matched controls. Neither genotype nor haplotype frequencies differed between cases and controls. However, the odds of developing basal cell carcinoma on the trunk compared to the head/neck appeared somewhat lower with carriers of the c.3298GC than the CC genotype. There was no evidence for interactions between skin type, childhood sunburning, average adult sun exposure, adult sunbathing, or intermittency of sun exposure and GLI1 haplotype. Additionally, we found no significant differences in transcription activation or cell transforming ability among the four GLI1 haplotypes. CONCLUSION: These results suggest that different GLI1 genotypes alone or in combination with past sun exposure patterns as assessed in this study do not affect basal cell carcinoma risk.

Application of small molecule CHIR99021 leads to the loss of hemangioblast progenitor and increased hematopoiesis of human pluripotent stem cells.

Improving our understanding of the intricacies of hematopoietic specification of induced or embryonic human pluripotent stem cells is beneficial for many areas of research and translational medicine. Currently, it is not clear whether, during human pluripotent stem cells hematopoietic differentiation in vitro, the maturation of definitive progenitors proceeds through a primitive progenitor (hemangioblast) intermediate or if it develops independently. The objective of this study was to investigate the early stages of hematopoietic specification of pluripotent stem cells in vitro. By implementing an adherent culture, serum-free differentiation system that utilizes a small molecule, CHIR99021, to induce human pluripotent stem cells toward various hematopoietic lineages, we established that, compared with the OP9 coculture hematopoietic induction system, the application of CHIR99021 alters the early steps of hematopoiesis such as hemangioblasts, angiogenic hematopoietic progenitors, and hemogenic endothelium. Importantly, it is associated with the loss of hemangioblast progenitors, loss of CD43+ (primitive hematopoietic marker) expression, and predominant development of blast-forming unit erythroid colonies in semisolid medium. These data support the hypothesis that the divergence of primitive and definitive programs during human pluripotent stem cells differentiation precedes the hemangioblast stage. Furthermore, we have shown that the inhibition of primitive hematopoiesis is associated with an increase in hematopoietic potential, which is a fruitful finding due to the growing need for lymphoid and myeloid cells in translational applications.

Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential.

BACKGROUND: The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis. METHODS: The hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential. RESULTS: Monolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31+CD34+ hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T-lymphoid, and B-lymphoid cells. CONCLUSION: The results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction, offers a defined system to study the factors that affect the early stages of hematopoiesis, and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells, thus enhancing their use in translational medicine.

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.

Factors required during preculture of rat oocytes soon after sperm penetration for promoting their further development in a chemically defined medium.

Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium.

Emerging roles for hedgehog-patched-Gli signal transduction in reproduction.

Hedgehog (Hh) proteins are expressed during vertebrate development in some tissues with inductive properties and at epithelial-mesenchymal boundaries in several developing organs, including the lung, gut, hair follicle, and tooth. The Hh signaling pathway is highly conserved, and important clues to understanding the mechanism of Hh signal transduction in vertebrates have come from studies in Drosophila. In recent years, Hh signaling has been recognized during embryonic development and in some cases during postnatal life in several mammalian tissues whose functions are essential for reproduction, including the gonads, uterus, and hormonally responsive accessory sex glands such as the prostate and mammary gland. The role of the pathway in these tissues is highly reminiscent of its role at epithelial-mesenchymal-stromal boundaries in other organ systems, which has provided a framework within which to explore Hh signaling in tissues that function in reproduction. Some features unique to these tissues are emerging, including a role in proliferation and differentiation of male germline cells in mammals and apparent influences of sex steroids on Hh signaling. However, many questions remain about the function of Hh signaling in the gonads, uterus, prostate, and mammary gland, including factors regulating the signal transduction pathway, identification of downstream target genes, and roles for Hh signaling in diseases involving these tissues.

Cooperative E-box regulation of human GLI1 by TWIST and USF.

Sonic hedgehog signaling plays a critical role in vertebrate patterning, and signaling defects are associated with severe birth defects and cancer in man. GLI1 encodes a critical transcription activator in this pathway. GLI1 is expressed in human basal cell carcinomas and sarcomas. Despite the significance of the GLI1 gene in human disease, few immediate upstream regulators of GLI1 expression are known. We previously demonstrated that a 5' region, including 5' flanking sequence, an untranslated exon, and 425 bp of the first intron, regulates the human GLI1 gene. Here we show that inactivating mutations in E-box, GC box, AP-2, GATA, GSG, PuF, and Zeste sites identified three critical regulatory elements, including a GC box that binds Sp1 and two intronic E-boxes that bind USF proteins or Twist. Expression of Twist but not a frame shift mutation of Twist activates the wild-type human GLI1 regulatory sequences but not with inactivating mutations of the E-boxes. Twist activates GLI1 reporter expression through E-box +482 but requires binding of USF proteins to E-box +157. Twist mutations cause human birth defects and Twist is overexpressed in many rhabdomyosarcomas, suggesting that one of Twist's primary roles is the regulation of GLI1.