Biomarkers and mechanisms of FANCD2 function.

Genetic or epigenetic inactivation of the pathway formed by the Fanconi anemia (FA) and BRCA1 proteins occurs in several cancer types, making the affected tumors potentially hypersensitive to DNA cross-linkers and other chemotherapeutic agents. It has been proposed that the inability of FA/BRCA-defective cells to form subnuclear foci of effector proteins, such as FANCD2, can be used as a biomarker to aid individualization of chemotherapy. We show that FANCD2 inactivation not only renders cells sensitive to cross-links, but also oxidative stress, a common effect of cancer therapeutics. Oxidative stress sensitivity does not correlate with FANCD2 or RAD51 foci formation, but associates with increased gammaH2AX foci levels and apoptosis. Therefore, FANCD2 may protect cells against cross-links and oxidative stress through distinct mechanisms, consistent with the growing notion that the pathway is not linear. Our data emphasize the need for multiple biomarkers, such as gammaH2AX, FANCD2, and RAD51, to capture all pathway activities.

Recombinant HoxB4 fusion proteins enhance hematopoietic differentiation of human embryonic stem cells.

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.

Scalable generation of universal platelets from human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the ß2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Hemangioblasts from human embryonic stem cells generate multilayered blood vessels with functional smooth muscle cells.

BACKGROUND: The formation and regeneration of functional vasculatures require both endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Identification and isolation of progenitors with potential for both EC and SMC lineage differentiation from an inexhaustible source, such as human embryonic stem (hES) or induced pluripotent stem cells, will be desirable for cell replacement therapy. METHOD: Recently, we have developed a serum-free and animal feeder-free differentiation system to generate blast cells (BCs) from hESCs. These cells possess the characteristics of hemangioblasts in vitro and are capable of repairing damaged retinal vasculatures, restoring blood flow in hind-limb ischemia and reducing the mortality rate after myocardial infarction in vivo. We demonstrate here that BCs express markers of SMCs and differentiate into smooth muscle-like cells (SMLCs), in addition to ECs and hematopoietic cells. RESULTS: When BCs from individual blast colonies were cultured in SMC medium, they differentiated into both ECs and SMLCs, which formed capillary-vascular-like structures after replating on Matrigeltrade mark. The SMLCs expressed SMC-specific markers (alpha-SM actin and calponin) and contracted upon treatment with carbachol. When implanted in nude mice, these cells formed microvasculature with ECs in Matrigel plaques. The BCs differentiated into both ECs and SMLCs, and incorporated into blood vessels after injection into ischemic tissue. CONCLUSION: These results demonstrate that hemangioblasts (BCs) generated from hESCs are tripotential and can provide a potentially inexhaustible source of cells for the treatment of human blood and vascular diseases.

Utility of DNA repair protein foci for the detection of putative BRCA1 pathway defects in breast cancer biopsies.

The DNA damage response pathway controlled by the breast cancer and Fanconi anemia (FA) genes can be disrupted by genetic or epigenetic mechanisms in breast cancer. Defects in this pathway may render the affected tumors hypersensitive to DNA-damaging agents. The identification of these defects poses a challenge because of the large number of genes involved in the FA/BRCA pathway. Many pathway components form subnuclear repair protein foci upon exposure to ionizing radiation in vitro, but it was unknown whether foci can be detected in live cancer tissues. Thus, the goal of this pilot study was to identify pathway defects by using a novel ex vivo foci biomarker assay on tumor biopsies. Fresh pretreatment biopsy specimens from patients with locally advanced sporadic breast cancer were irradiated or mock-treated in the laboratory (ex vivo). Foci formation of DNA repair proteins BRCA1, FANCD2, and RAD51 was detected by immunofluorescence microscopy. Three out of seven tumors showed intact radiation-induced foci formation, whereas the other four tumors exhibited a defective foci response. Notably, three of the foci-defective tumors were estrogen receptor/progesterone receptor/HER2-negative (triple-negative), a phenotype that has been associated with BRCA1 deficiency. In conclusion, in this pilot study, we report the successful detection of BRCA1, FANCD2, and RAD51 foci in breast cancer biopsies irradiated ex vivo. Our approach represents a potentially powerful biomarker assay for the detection of pre-existing and functionally important defects within the complex FA/BRCA pathway, which may ultimately allow us to tailor cancer treatment to the DNA repair profile of individual tumors.

Robust generation of hemangioblastic progenitors from human embryonic stem cells.

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However, successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models, the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary, we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury, KDR, CD31 and Lmo2 gene expression, while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development, consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.