Generation of cryopreserved macrophages from normal and genetically engineered human pluripotent stem cells for disease modelling.

Macrophages are innate immune cells that play critical roles in tissue homeostasis, inflammation, and immune oncology. Macrophages differentiated from human induced pluripotent stem cells (iPSCs) overcome many limitations of using peripheral blood derived macrophages. The ability to scale up and cryopreserve a large amount of end stage macrophages from single clonal iPSCs from normal and disease specific donors offers a unique opportunity for genomic analysis and drug screening.

iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery.

Non-alcoholic fatty liver disease (NAFLD) affects 30-40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets.

Karenitecin (bnp1350) and flavopridol as radiosensitizers in malignant glioma.

The poor prognosis of malignant glioma patients highlights the need to develop low toxicity, tumor specific agents with the ability to synergize with proven efficacious treatment modalities, e.g., ionizing irradiation.

Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors.

The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood.

Analysis of the CD1 antigen presenting system in humanized SCID mice.

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34(+) hematopoietic stem cells develop a functional human CD1 compartment.

Human lymphoblastoid B-cell lines reprogrammed to EBV-free induced pluripotent stem cells.

Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules.

A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system.

A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas.

Epstein-Barr virus (EBV) infects cells in latent or lytic forms, but the role of lytic infection in EBV-induced lymphomas is unclear. Here, we have used a new humanized mouse model, in which both human fetal CD34(+) hematopoietic stem cells and thymus/liver tissue are transplanted, to compare EBV pathogenesis and lymphoma formation following infection with a lytic replication-defective BZLF1-deleted (Z-KO) virus or a lytically active BZLF1(+) control. Both the control and Z-KO viruses established long-term viral latency in all infected animals.

Derivation of induced pluripotent stem cells from human peripheral blood T lymphocytes.

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs ("TiPS") retain a normal karyotype and genetic identity to the donor.

Th1 and Th17 immunocompetence in humanized NOD/SCID/IL2rgammanull mice.

We evaluated the immunocompetence of human T cells in humanized NOD-SCID interleukin (IL)-2r-gamma-null (hu-NSG) mice bearing a human thymic organoid, after multilineage reconstitution with isogeneic human leukocytes. Delayed type hypersensitivity (DTH) response was assessed by a direct footpad challenge of the immunized hu-NSG host, or by transfer of splenocytes from immunized hu-NSG, along with antigen, into footpads of C.B-17 scid mice (trans vivo [tv] DTH).

Early chimerism threshold predicts sustained engraftment and NK-cell tolerance in prenatal allogeneic chimeras.

The failure of engraftment in human cases of in utero hematopoietic cell transplantation (IUHCT) in which no immunodeficiency exists suggests the presence of an unrecognized fetal immune barrier. A similar barrier in murine IUHCT appears to be dependent on the chimerism level and is poorly explained by a lack of T-cell tolerance induction. Therefore, we studied the effect of the chimerism level on engraftment and host natural killer (NK)-cell education in a murine model of IUHCT.

Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells.

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs.

Motexafin-gadolinium taken up in vitro by at least 90% of glioblastoma cell nuclei.

Purpose: We present preclinical data showing the in vitro intranuclear uptake of motexafin gadolinium by glioblastoma multiforme cells, which could serve as a prelude to the future development of radiosensitizing techniques, such as gadolinium synchrotron stereotactic radiotherapy (GdSSR), a new putative treatment for glioblastoma multiforme.

Experimental Design: In this approach, administration of a tumor-seeking Gd-containing compound would be followed by stereotactic external beam radiotherapy with 51-keV photons from a synchrotron source. At least two criteria must be satisfied before this therapy can be established: Gd must accumulate in cancer cells and spare the normal tissue; Gd must be present in almost all the cancer cell nuclei.

Are gadolinium contrast agents suitable for gadolinium neutron capture therapy?

Objective: Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific (157)Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents.

Perillyl alcohol mediated radiosensitization via augmentation of the Fas pathway in prostate cancer cells.

Background: The management of hormone-insensitive locally advanced prostate cancer is difficult and complex and there is an urgent need for the development of effective chemotherapeutic agents intended for combination with currently available treatment modalities.

Methods: The present paper demonstrates the effectiveness of the monoterpene perillyl alcohol (POH) as potent radiosensitizer on DU145 and PC3 cell lines by performing clonogenic survival assays, cycle analysis, and assays to detect viability, apoptosis, and Fas receptor/ligand by flow cytometry.

Results: POH pretreatment resulted in a dose dependent sensitization to kill cell by radiation.

Perillyl alcohol as a radio-/chemosensitizer in malignant glioma.

The prognosis for patients with malignant glioma has not significantly changed in two decades, despite advances in surgery, radiation, and chemotherapy, emphasizing the growing need for novel approaches to glioma therapy. Perillyl alcohol (POH) is a naturally occurring monoterpene that has been shown to possess chemotherapeutic as well as chemopreventive activity in animal tumor models and is currently in Phase I and Phase II clinical trials. In the present study, we have demonstrated that POH is an effective radiosensitizer at clinically relevant doses of radiation using established glioma cell lines.