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.

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.

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.

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.

Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma.

BACKGROUND: The clinical significance of GLI1 expression either through canonical Hedgehog signal transduction or through non-canonical mechanisms in rhabdomyosarcoma (RMS) or Ewing sarcoma (EWS) is incompletely understood. We tested a role for Hedgehog (HH) signal transduction and GL11 expression in development of vincristine (VCR) resistance in RMS and EWS. METHODS: We characterized baseline expression and activity of HH pathway components in 5 RMS (RD, Rh18, Ruch-2, Rh30, and Rh41) and 5 EWS (CHLA9, CHLA10, TC32, CHLA258, and TC71) cell lines. We then established VCR-resistant RMS and EWS cell lines by exposing cells to serially increasing concentrations of VCR and determining the IC50. We defined resistance as a ≥ 30-fold increase in IC50 compared with parental cells. We determined changes in gene expression in the VCR-resistant cells compared with parental cells using an 86-gene cancer drug resistance array that included GLI1 and tested the effect of GLI1 inhibition with GANT61 or GLI1 siRNA on VCR resistance. RESULTS: We found evidence for HH pathway activity and GLI1 expression in RMS and EWS cell lines at baseline, and evidence that GLI1 contributes to survival and proliferation of these sarcoma cells. We were able to establish 4 VCR-resistant cell lines (Ruch-2VR, Rh30VR, Rh41VR, and TC71VR). GLI1 was significantly up-regulated in the Rh30VR, Rh41VR, and TC71VR cells. The only other gene in the drug resistance panel that was significantly up-regulated in each of these VCR-resistant cell lines compared with their corresponding parental cells was the GLI1 direct target and multidrug resistance gene, ATP-binding cassette sub-family B member 1 (MDR1). We established major vault protein (MVP), which was up-regulated in both vincristine-resistant alveolar RMS cell lines (Rh30VR and Rh41VR), as another direct target of GLI1 during development of drug resistance. Treatment of the VCR-resistant cell lines with the small molecule inhibitor GANT61 or GLI1 siRNA together with VCR significantly decreased cell viability at doses that did not reduce viability individually. CONCLUSIONS: These experiments demonstrate that GLI1 up-regulation contributes to VCR resistance in RMS and EWS cell lines and suggest that targeting GLI1 may benefit patients with RMS or EWS by reducing multidrug resistance.

Regulation of GLI1 by cis DNA elements and epigenetic marks.

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5' exons and large introns 5' of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

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.

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.

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.

p53 modulates the activity of the GLI1 oncogene through interactions with the shared coactivator TAF9.

The GLI1 oncogene and p53 tumor suppressor gene function in an inhibitory loop that controls stem cell and tumor cell numbers. Since GLI1 and p53 both interact with the coactivator TATA Binding Protein Associated Factor 9 (TAF9), we hypothesized that competition between these transcription factors for TAF9 in cancer cells may contribute to the inhibitory loop and directly affect GLI1 function and cellular phenotype. We showed that TAF9 interacts with the oncogenic GLI family members GLI1 and GLI2 but not GLI3 in cell-free pull-down assays and with GLI1 in rhabdomyosarcoma and osteosarcoma cell lines. Removal of the TAF9-binding acidic alpha helical transactivation domain of GLI1 produced a significant reduction in the ability of GLI1 to transform cells. We then introduced a point mutation into GLI1 (L1052I) that eliminates TAF9 binding and a point mutation into GLI3 (I1510L) that establishes binding. Wild-type and mutant GLI proteins that bind TAF9 showed enhanced transactivating and cell transforming activity compared with those that did not. Therefore, GLI-TAF9 binding appears important for oncogenic activity. We then determined whether wild-type p53 down-regulates GLI function by sequestering TAF9. We showed that p53 binds TAF9 with greater affinity than does GLI1 and that co-expression of p53 with GLI1 or GLI2 down-regulated GLI-induced transactivation, which could be abrogated using mutant forms of GLI1 or p53. This suggests that p53 sequesters TAF9 from GLI1, which may contribute to inhibition of GLI1 activity by p53 and potentially impact therapeutic success of agents targeting GLI-TAF9 interactions in cancer.

Promoting convergence: the Phi spiral in abduction of mouse corneal behaviors.

Why do mouse corneal epithelial cells display spiraling patterns? We want to provide an explanation for this phenomenon by applying an idealized problem solving process. Specifically, we applied complementary line-fitting methods to measure transgenic epithelial reporter expression arrangements displayed on three mature, live enucleated globes to clarify the problem. Two prominent logarithmic curves were discovered, one of which displayed the ϕ ratio, an indicator of the optimal configuration in phyllotactic systems. We then utilized two different computational approaches to expose our current understanding of the behavior. In one procedure, which involved an isotropic mechanics-based finite element method, we successfully produced logarithmic spiral curves of maximum shear strain based pathlines but computed dimensions displayed pitch angles of 35° (ϕ spiral is ~17°), which was altered when we fitted the model with published measurements of coarse collagen orientations. We then used model-based reasoning in context of Peircean abduction to select a working hypothesis. Our work serves as a concise example of applying a scientific habit of mind and illustrates nuances of executing a common method to doing integrative science.

Noncanonical regulation of the Hedgehog mediator GLI1 by c-MYC in Burkitt lymphoma.

Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1, and SMO and that five of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO, and SuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 5'-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box-dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These findings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers.

Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation.

BACKGROUND: Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh) signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. RESULTS: In the presence of exogenous ascorbic acid (AA), a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells) formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis inhibitor 3,4-dehydro-L-proline. CONCLUSIONS: Matrix collagen, formed by osteoblasts in the presence of AA, potentiates Shh signaling between Shh-expressing prostate cancer cells and osteoblasts. Collagen and Shh signaling exert a synergistic effect on osteoblast differentiation, a defining event in prostate carcinoma bone metastasis. Investigations into paracrine interactions among prostate cancer cells, osteoblasts, and osteoblast-synthesized matrix proteins advance our understanding of mechanisms contributing to prostate cancer bone metastasis.

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.

Three dimensional visualization and fractal analysis of mosaic patches in rat chimeras: cell assortment in liver, adrenal cortex and cornea.

The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations.