Search

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

Cellular therapies combined with genetic engineering technologies have emerged as increasingly powerful tools for immunotherapies of cancers and viral infections including HIV infection. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of blood progenitors and terminally differentiated cells can further expand applicability of cellular immunotherapies by offering “off-the-shelf” therapeutic products to fit specific clinical needs for a broad group of patients. In this review, we discuss current advances in lymphoid and myeloid cell differentiation of iPSCs along with challenges in de novo generation of hematopoietic stem cells (HSCs) and allogeneic iPSC products for “off-the-shelf” immunotherapies.

Adoptive therapies with genetically modified somatic T cells rendered HIV resistant have shown promise for AIDS therapy. A renewable source of HIV resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from peripheral blood T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian Cynomolgus macaques (MCM), using CRISPR/Cas9 technology. We found that CCR5 editing does not affect pluripotency or hematopoietic and T cell differentiation potentials of fib-iPSCs. However, deletion of CCR5 in T-iPSCs leads to selective loss of their T cell redifferentiation potential without affecting myeloid development. T cells and macrophages produced from CCR5-edited MCM- iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T-cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate preclinical model.

BackgroundAllogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood but may include pre-transplant conditioning regimens, ART-mediated protection of donor cells, and graft-versus-host (GvH) responses. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay.ResultsWe infected four MCMs with CCR5-tropic SHIV162P3 and started ART 6-16 weeks post-infection (p.i.) to establish robust viral reservoirs. We maintained the MCMs on continuous ART during myeloablative conditioning with total body irradiation (TBI) and while transplanting allogeneic MHC-matched α/β T cell-depleted bone marrow cells. Post-transplant, we prophylactically treated the MCMs with cyclophosphamide and tacrolimus to prevent GvH disease (GvHD). The transplants produced ~85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their peripheral blood mononuclear cells post-transplant. However, SHIV-harboring cells persisted in various tissues. We detected viral DNA in lymph node biopsies and terminal analyses of tissues between 38 and 62 days post-transplant. Further, we removed ART from one MCM at 63 days post-transplant, resulting in viral rebound within seven days and viral loads nearing 1×108SHIV RNA copies/ml of plasma after treatment interruption.ConclusionsOur results indicate that myeloablative conditioning and maintaining ART through the peri-transplant period alone are insufficient for eradicating latent viral reservoirs early after allo-HSCTs. Furthermore, our findings suggest that extended ART and GvH responses may be necessary to substantially deplete viral reservoirs after allo-HSCTs.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models. Here we evaluated the outcomes of major histocompatibility complex-matched T-cell receptor α/β-depleted alloHSCT in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/β depletion, bone marrow cells were transplanted into major histocompatibility complex-identical MCMs conditioned with total body irradiation. GvHD prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Posttransplant chimerism was determined by sequencing diagnostic single-nucleotide polymorphisms to quantify the amounts of donor and recipient cells present in blood. Animals treated posttransplant with sirolimus developed nearly complete chimerism with acute GvHD. In the cyclophosphamide and tacrolimus treatment group, animals developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. None of the animals developed cytomegalovirus infection. These studies indicate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. Further exploration of this model will provide a platform for elucidating the mechanisms of GvHD and GvHE and for testing novel alloHSCT modalities for HIV infection.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

Specification of endothelial cells (ECs) into arterial, venous, and lymphatic cells is a crucial process of vascular development, and expanding our knowledge about EC specification from human pluripotent stem cells (hPSCs) will aid the design of optimal strategies for producing desired types of ECs for therapies. In our prior studies, we revealed that hPSC-derived VE-cadherin(V)(+)CD31(+)CD34(+) ECs are heterogeneous and include at least three major subsets with distinct hemogenic properties: V(+)CD43/235a(−)CD73(−) hemogenic endothelial progenitors (HEPs), V(+)CD43(lo)CD235a(+)73(−) angiogenic hematopoietic progenitors (AHPs), and V(+)CD43/235a(−)73(+) non-HEPs. In this study, using angiogenesis assays, we demonstrated that ECs within these subsets have distinct endothelial colony- and tube-forming properties, proliferative and migratory properties, and endothelial nitric oxide synthase and inflammatory cytokine production potentials. Culture of isolated subsets in arterial, venous, and lymphatic conditions revealed that AHPs are skewed toward lymphatic, HEPs toward arterial, and non-HEPs toward venous differentiation in vitro. These findings suggest that selection and enhancement of production of a particular EC subset may aid in generating desirable EC populations with arterial, venous, or lymphatic properties from hPSCs.

Bone marrow suppression and associated neutropenia that renders the patient more susceptible to infection is a major complication and limiting factor for current chemotherapy. Strategies aimed at neutrophil reconstitution using transfusions or accelerating neutrophil recovery with G-CSF show a limited effect in reducing the rate of infection. To overcome these limitations, administration of ex vivo expanded somatic hematopoietic progenitors (HPs) has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival following myeloablation. Recent advances in reprogramming and induced pluripotent stem cell (iPSC) technologies have created alternative platforms for off-the-shelf supply of immunologically compatible HPs, including cellular products derived from MHC homozygous superdonors which can increase the degree of MHC matching and may provide a maximum utility for stem cell banking. In these studies, we developed Mauritian cynomolgus macaque (MCM) model to evaluate the utility and safety of CD34 +CD45 + hematopoietic progenitors derived from iPSCs (iHPs) generated from MHC homozygous animals in the treatment of cytopenia following myeloablative stem cell transplantation. MCM iPSCs were generated from MHC homozygous animals and used to generate iHPs in coculture with OP9. Three groups of MCMs underwent myeloablative total body irradiation (TBI) followed by transplantation of cryopreserved autologous CD34 + HSPCs. Four days after autologous HSPC transplantation, animals received transfusion of 30x10 6/kg cryopreserved iCD34 + cells tagged with eGFP or tdTomato from homozygous MHC-matched iPSCs (group 2) or homozygous MHC mismatched iPSCs (group 3). We have demonstrated that infusion of iHPs is safe and well tolerated. No teratoma or tumor formation was observed in animals one year after infusion of iHPs. Although we detected few iPSC derived hematopoietic cells in the blood within 1-week post-infusion, we did not see any significant differences in blood counts or other peripheral blood parameters between all animal groups. Intriguingly we found footprints of iPSC-derived cells in the colon, lymph node, skin and brain specimens collected 25 days after iHP infusion from one animal. This suggests a possibility of migration and retention within these tissues of iHP and iHP-derived myeloid cells. To assess immunogenicity, MHC homozygous iHPs were infused into immunocompetent MCMs across different MHC barriers. 10x10 6/kg of cryopreserved iCD34+ from homozygous iPSCs were transfused every week for 3 weeks, into animals that were divided into 4 groups namely autologous (group I), MHC matched homozygous (group II), MHC matched heterozygous (group III) and MHC mismatch (group IV). Overall, these studies revealed low immunogenicity of MHC homozygous iHPs in MHC matched homozygous and heterozygous animals, while weak immune response was detected following iHP infusion in some MHC mismatched animals. Disclosures Slukvin: Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.

SUMMARY Elucidating the pathways that lead to vasculogenic cells, and being able to identify their progenitors and lineage-restricted cells, is critical to the establishment of human pluripotent stem cell (hPSC) models for vascular diseases and development of vascular therapies. Here, we find that mesoderm-derived pericytes (PCs) and smooth muscle cells (SMCs) originate from a clonal mesenchymal progenitor mesenchymoangioblast (MB). In clonogenic cultures, MBs differentiate into primitive PDGFRβ+ CD271+CD73− mesenchymal progenitors, which give rise to proliferative PCs, SMCs, and mesenchymal stem/stromal cells. MB-derived PCs can be further specified to CD274+ capillary and DLK1+ arteriolar PCs with a proinflammatory and contractile phenotype, respectively. SMC maturation was induced using a MEK inhibitor. Establishing the vasculogenic lineage tree, along with identification of stage- and lineage-specific markers, provides a platform for interrogating the molecular mechanisms that regulate vasculogenic cell specification and diversification and manufacturing well-defined mural cell populations for vascular engineering and cellular therapies from hPSCs., In Brief Kumar et al. find that mesodermal pericytes and smooth muscle cells in human pluripotent stem cell cultures originate from a common endothelial and mesenchymal cell precursor, the mesenchymoangioblast. They show how different lineages of mural cells are specified from mesenchymoangioblasts and define stage- and lineage-specific markers for vasculogenic cells., Graphical Abstract

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

Hematopoietic stem cells (HSCs) surface during embryogenesis leading to the genesis of the hematopoietic system, which is vital for immune function, homeostasis balance, and inflammatory responses in the human body. Hematopoiesis is the process of blood cell formation, which initiates from hematopoietic stem/progenitor cells (HSPCs) and is responsible for the generation of all adult blood cells. With their self-renewing and pluripotent properties, human pluripotent stem cells (hPSCs) provide an unprecedented opportunity to createin vitromodels of differentiation that will revolutionize our understanding of human development, especially of the human blood system. The utilization of hPSCs provides newfound approaches for studying the origins of human blood cell diseases and generating progenitor populations for cell-based treatments. Current shortages in our knowledge of adult HSCs and the molecular mechanisms that control hematopoietic development in physiological and pathological conditions can be resolved with better understanding of the regulatory networks involved in hematopoiesis, their impact on gene expression, and further enhance our ability to develop novel strategies of clinical importance. In this review, we delve into the recent advances in the understanding of the various cellular and molecular pathways that lead to blood development from hPSCs and examine the current knowledge of human hematopoietic development. We also review howin vitrodifferentiation of hPSCs can undergo hematopoietic transition and specification, including major subtypes, and consider techniques and protocols that facilitate the generation of hematopoietic stem cells.

Identification of transcription factors that directly convert pluripotent stem cells (PSCs) into endothelial and blood cells and advances in the chemical modifications of messenger RNA (mRNA) offer alternative nucleic acid-based transgene-free approach for scalable production of these cells for drug screening and therapeutic purposes. Here we evaluated the effect of 5′ and 3′ RNA untranslated regions (UTRs) on translational efficiency of chemically-modified synthetic mRNA (modRNA) in human PSCs and showed that an addition of 5′UTR indeed enhanced protein expression. With the optimized modRNAs expressing ETV2 or ETV2 and GATA2, we are able to produce VE-cadherin(+) endothelial cells and CD34(+)CD43(+) hematopoietic progenitors, respectively, from human PSCs as well as non-human primate (NHP) PSCs. Overall, our findings provide valuable information on the design of in vitro transcription templates being used in PSCs and its broad applicability for basic research, disease modeling, and regenerative medicine.

Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs. To improve T cell production from hPSCs, it is essential to identify cell subsets that are highly enriched in T cell progenitors and those stages of development at which NOTCH activation induces the most potent T cells. In this study, we evaluated the efficacy of T cell production from cell populations isolated at different stages of hematopoietic differentiation, including mesoderm, hemogenic endothelium (HE), and multipotent hematopoietic progenitors. We demonstrate that KDRhiCD31− hematovascular mesodermal progenitors (HVMPs) with definitive hematopoietic potential produce the highest numbers of T cells when cultured on OP9-DLL4 as compared with downstream progenitors, including HE and multipotent hematopoietic progenitors. In addition, we found that T cells generated from HVMPs have the capacity to expand for 6–7 wk in vitro, in comparison with T cells generated from HE and hematopoietic progenitors, which could only be expanded for 4–5 wk. Demonstrating the critical need of NOTCH activation at the HVMP stage of hematopoietic development to establish robust T cell production from hPSCs may aid in establishing protocols for the efficient off-the-shelf production and expansion of T cells for treating hematologic malignancies.

The angiopoietin/Tie-2 system has been identified as a key role player in tumor angiogenesis. We investigated whether angiopoietin-2 could be a promising target in human neuroblastoma.Angiopoietin-2 down-regulation by siRNA or shRNA was evaluated in vitro in Kelly cells. Angiopoietin-2 shRNA-transfected Kelly cells were tested in a chorioallantoic membrane (CAM) assay to evaluate tumor growth and microvessel density. The effects of L1-10, a peptide-Fc fusion molecule blocking angiopoietin-2/Tie-2 interaction, administered 3 times/week were assessed in a murine neuroblastoma xenograft model.Angiopoietin-2 down-regulation by siRNA or shRNA in Kelly cells inhibited cell proliferation and migration. In vivo growth and microvessel density of angiopoietin-2 shRNA-transfected Kelly cells in the CAM assay were reduced. Therapy of advanced tumors with L1-10 did not stop tumor progression. However, starting L1-10 treatment at the same time as neuroblastoma cell injection significantly inhibited tumor growth (vehicule: 903 ± 160 mm(3); L1-10: 270 ± 152 mm(3) after 26 days; P 0.05). Microvessel density was reduced in both L1-10-treated tumors, whereas expression of angiopoietin-2 and VEGF-A did not change.This first demonstration of beneficial angiopoietin-2 inhibition in neuroblastoma offers an additional approach for future therapy strategies, especially by using L1-10 in the setting of minimal residual disease.

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

1,3,4-Oxadiazoles are an important class of heterocyclic compounds, which play a pivotal role in various pharmaceutical applications. Here, we investigated the antiangiogenic and antiproliferative effects of the derivatives and explored its mechanism of action on EAT cells.The cytotoxic effect of the derivatives on EAT and HEK293 cells was assessed by MTT assay. Effect of the derivatives on ALP activity and proliferation was measured. Swiss albino mice transplanted with EAT cells were used as a model system to study the effect of the derivatives in vivo. Inhibition of angiogenesis in mice peritoneum, CAM and Cornea of the rat were studied. Finally, the effects on VEGF gene expression, HIF-1alpha translocation and cell cycle arrest were determined.The IC50 range for growth inhibition of EAT cells was found to be 140-175 microM. In contrast normal HEK293 cells were resistant to the derivatives at this range. Treatment with derivatives in vivo was demonstrated by the down regulation of VEGF in EAT cells and inhibition of blood vessels formation in mice peritoneum, CAM and cornea of rat, indicating the potent angioinhibitory effect of the derivatives. VEGF promoter-luciferase reporter gene expression analysis showed suppression of VEGF gene expression in vitro. The derivatives proved to be potent antiproliferative agents as shown by FACS analysis and decreased ALP activity. Furthermore, expression of HIF-1alpha was also down regulated by derivatives by repressing its nuclear translocation.Oxadiazole derivatives are strong bioactive compounds with antiangiogenic and antiproliferative potential both in vitro and in vivo. We postulate that diminished HIF-1alpha nuclear presence in oxadiazole treated EAT cells could be responsible for decreased VEGF expression and antiangiogenic effects.

The authors investigate the antiangiogenic and proapoptotic effects of mustard essential oil containing allyl isothiocyanate (AITC) and explore its mechanism of action on Ehrlich ascites tumor (EAT) cells. Swiss albino mice transplanted with EAT cells were used to study the effect of AITC. AITC was effective at a concentration of 10 μm as demonstrated by the inhibition of proliferation of EAT cells when compared with the normal HEK293 cells. It significantly reduced ascites secretion and tumor cell proliferation by about 80% and inhibited vascular endothelial growth factor expression in tumor-bearing mice in vivo. It also reduced vessel sprouting and exhibited potent antiangiogenic activity in the chorioallantoic membrane and cornea of the rat. AITC arrested the growth of EAT cells by inducing apoptosis and effectively arrested cell cycle progression at the G1 phase. The results clearly suggest that AITC inhibits tumor growth by both antiangiogenic and proapoptotic mechanisms.

Background aims Existing treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. Methods Induced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 10 6 cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. Results Blood flow in the MCA-derived MSC-treated animals was higher at each day ( P 0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P 0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. Discussion MCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.

A major obstacle to the success of gene therapy for hematologic disorders is the inefficiency of lentiviral vector (LV) gene transfer to hematopoietic stem cells (HSCs). Achieving clinically relevant gene delivery levels requires prolonged ex vivo culture of HSCs with cytokines and large LV doses. Rapamycin, PGE2, and other small molecules, have been reported to increase LV transduction of HSCs, however the mechanism of action of these drugs and the basis for LV restriction in HSCs are poorly understood.Here, we report a novel small molecule, β-deliverin, which improves LV gene transfer to HSCs up to 3 fold over DMSO control in vitro. This effect is most pronounced in human adult peripheral blood-derived HSCs, but also observed in human cord-derived HSCs, and in non-human primate bone marrow-derived HSCs. Importantly, treatment with β-deliverin does not significantly affect the viability or expansion of HSCs in culture. Furthermore, for cord-derived HSCs, there was no reduction in the number or type of colony-forming cells. In vivo, β-deliverin treated HSCs engraft in the peripheral blood of NSG mice at levels comparable to control at 16 weeks post-engraftment. Despite an only modest increase in transduction efficiency in β-deliverin-treated cord-derived HSCs transduced in vitro (26% versus 20% for control), the marking frequency for control cells dropped to

Induced pluripotent stem cells (iPSCs) have created novel opportunities for the scalable manufacture of blood products for clinical use. Recent advances in hematopoietic differentiation from human pluripotent stem cells have brought the clinical translation of iPSC-derived blood products close to reality and evoke an urgent need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models. Due to substantial similarities with humans, outcomes of cellular therapies in nonhuman primate (NHP) models can be readily extrapolated to clinical setting. However, the use of this model is hampered by limited availability of clinically relevant NHP-iPSCs and low efficiency of myeloid and lymphoid differentiation from them. Here, we describe generation of clinically relevant transgene-free iPSCs from different NHP species, including rhesus, Chinese and Mauritian cynomolgus monkeys, and demonstrate that GSK3β inhibition is essential to induce rapid and efficient differentiation of the NHP-iPSCs into multipotential CD34+CD45+CD90+CD38-CD45RA-hematopoietic progenitors in coculture with OP9 bone marrow stromal cells. NHP-iPSC-derived hematopoietic progenitors were capable of differentiating further into mature erythroid cells, megakaryocytes, granulocytes, monocytes/macrophages following exposure to hematopoietic cytokines promoting development corresponding lineages. In addition, iPSC-derived CD34+CD45+ cells generated CD4+CD8+ T lymphoid cells and CD159a+ NK cells when cultured on OP9 cells expressing DLL4. To confirm T cell development, we analyzed the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures for the presence of T cell receptor (TCR) rearrangements. This analysis demonstrated the presence of multiple PCR products of random V-J and D-J rearrangements at the β locus and V-J rearrangements at the γ locus, indicative of a polyclonal T lineage repertoire. To evaluate feasibility of using NHP model for evaluation of safety and clinical efficacy of iPSC-derived blood products, we generated iPSCs from Mauritian cynomolgus macaque and marked them with GFP. GFP-iPSCs were used to generate CD34+CD45+ multipotential hematopoietic progenitors using multiple rounds of differentiation followed by freezing. Subsequently, iPSC donor animal was subjected to nonmyeloablative conditioning with fludarabine (50 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and transfused with 30x106/kg autologous iPSC-derived CD34+CD45+ cells. Transfusion of iPSC-derived CD34+CD45+ cells well tolerated and animal recovered without any adverse events. By flow cytometry, GFP-marked cells (up to 1%) were detected in peripheral blood on days 20-30 post-transfusion, predominately in granulocyte, monocyte and platelet compartments. Although, GFP-positive cells mostly disappeared in circulation one month after transfusion, GFP-positive cells were detected in circulation for more than 90 days by genomic PCR. During 6 month follow-up period no signs of myelo- or lympho-proliferative disorder were detected. Overall, these studies lay the foundation for advancing a NHP model for preclinical testing of iPSC-based therapies for blood diseases. Disclosures Slukvin: Cynata: Consultancy, Equity Ownership, Research Funding.

Background Inhibition of tumor angiogenesis is a promising approach for cancer therapy and the Tie-2/angiopoietin pathway appears to play an important role. In the present study, we have developed strategies to explore the therapeutic potential of blocking the Tie-2/angiopoietin pathway by sTie-2. Methods Ehrlich ascites tumor (EAT) cells were stably transfected to overexpress a truncated form of sTie-2. Transfectants were characterized for their in vitro growth behavior and transplanted into nude mice. Furthermore, recombinant sTie-2 produced by the baculovirus expression system was used to sequester angiopoietins in the murine ascites carcinoma model. The effect of sTie-2 treatment alone or in combination with sFLT-1 on the weight of the animal, ascites cell number and volume was studied. Results EAT cells stably transfected with a truncated form of sTie-2 showed no change in cell proliferation in vitro and colony forming in soft agar compared to control cells. However, sTie-2 transfected EAT cells transplanted into nude mice reduced tumor burden and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Recombinant sTie-2 showed angiogenic activity in the tube formation and wound healing assay in vitro. sTie-2 treatment alone or in combination with sFLT-1 in an ascites tumor mouse model resulted in reduced peritoneal angiogenesis, with a concomitant decrease in tumor cell number, volume of ascites and the number of invasive tumor cells, as assayed by CD31 staining. Conclusions The findings of the present study demonstrate an important role for the Tie-2/angiopoietin pathway in the formation of tumor vasculature and suggest that sTie-2 might yield useful anticancer therapy. Copyright © 2010 John Wiley & Sons, Ltd.

The multiple pharmacological actions of unique synthetic compounds are a prerequisite for classifying a drug as highly efficacious, because the multiple pharmacological actions offer the possibility of treating various diseases like cancer. 1,3,4-Oxadiazoles are an important class of heterocyclic compounds with broad spectrum of biological activities. In this study we focused on the ability of these derivatives to induce apoptosis in cultured MCF-7 breast cancer cells. Treatment of MCF-7 cells with varying concentrations of the different derivatives resulted in dose and time dependent sequence of events marked by apoptosis, as shown by loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation and sub G(0) phase accumulation. Furthermore, apoptosis in MCF-7 cell was induced by upregulation of proto-oncoprotein Bax and activation of Caspase-3 activated DNase. Although the derivatives induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Analysis of the data suggests that the substituted oxadiazole derivatives exert antiproliferative action and growth inhibition on MCF-7 cells through apoptosis induction and that it may have anticancer properties valuable for application in drug products.