Viviane Tabar

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinsons disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinsons disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinsons disease.Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson’s disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson’s disease.

Glioblastoma stem-like cells give rise to tumour endothelium

Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133+ fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133+ stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133+/CD144+ progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133+ tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a γ-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133+ cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.

Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage

Neural stem cells (NSCs) yield both neuronal and glial progeny, but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast, embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs), a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate, we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals, R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition, the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs.

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest–related disorders.

Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.

Pluripotent stem cells in regenerative medicine: challenges and recent progress

After years of incremental progress, several recent studies have succeeded in deriving disease-relevant cell types from human pluripotent stem cell (hPSC) sources. The prospect of an unlimited cell source, combined with promising preclinical data, indicates that hPSC technology may be on the verge of clinical translation. In this Review, we discuss recent progress in directed differentiation, some of the new technologies that have facilitated the success of hPSC therapies and the remaining hurdles on the road towards developing hPSC-based cell therapies.

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell–derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell–derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell–derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.

Stereotactic Radiosurgery for Melanoma Brain Metastases in Patients Receiving Ipilimumab: Safety Profile and Efficacy of Combined Treatment

PURPOSE Ipilimumab (Ipi), a monoclonal antibody against cytotoxic T-lymphocyte antigen-4, has been shown to improve survival in patients with metastatic melanoma. In this single-institution study, we investigated the safety and efficacy of stereotactic radiosurgery (SRS) for patients with melanoma brain metastases (BMs) who also received Ipi. METHODS AND MATERIALS From 2005 to 2011, 46 patients with melanoma received Ipi and underwent single-fraction SRS for BMs. A total of 113 BMs (91% intact, 9% postoperative) were treated with a median dose of 21 Gy (range, 15-24 Gy). Ipi was given at 3 mg/kg (54%) or 10 mg/kg (46%) for a median of 4 doses (range, 1-21). Adverse events were recorded with the use of the Common Terminology Criteria for Adverse Events 3.0. Kaplan-Meier methods were used to estimate survival, and Cox regression was used to investigate associations. RESULTS Fifteen patients received SRS during Ipi, 19 received SRS before Ipi, and 12 received SRS after Ipi. Overall survival (OS) was significantly associated with the timing of SRS/Ipi (P=.035) and melanoma-specific graded prognostic assessment (P=.013). Patients treated with SRS during or before Ipi had better OS and less regional recurrence than did those treated with SRS after Ipi (1-year OS 65% vs 56% vs 40%, P=.008; 1-year regional recurrence 69% vs 64% vs 92%, P=.003). SRS during Ipi also yielded a trend toward less local recurrence than did SRS before or after Ipi (1-year local recurrence 0% vs 13% vs 11%, P=.21). On magnetic resonance imaging, an increase in BM diameter to >150% was seen in 50% of patients treated during or before Ipi but in only 13% of patients treated after Ipi. Grade 3 to 4 toxicities were seen in 20% of patients. CONCLUSION Overall, the combination of Ipi and SRS appears to be well tolerated. Concurrent delivery of Ipi and SRS is associated with favorable locoregional control and possibly longer survival. It may also cause a temporary increase in tumor size, possibly because of an enhanced immunomodulatory effect.

Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation

Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice. A stem cell model of a lethal brain tumor in children shows how a recurrent histone mutation leads to cancer. [Also see Perspective by Becher and Wechsler-Reya] Modeling brain cancer from stem to stern Diffuse intrinsic pontine gliomas (DIPGs) are aggressive brain tumors primarily affecting children. Because the tumors arise in the brainstem, which controls many vital functions, they cannot be surgically excised and are often fatal. To study the pathogenesis of DIPGs—in particular, the role of a histone H3.3 mutation that occurs in 70% of cases—Funato et al. developed a new tumor model (see the Perspective by Becher and Wechsler-Reya). They first directed the differentiation of embryonic stem cells into neural progenitor cells. They then introduced a specific combination of genes, including the mutant histone gene, and found that this caused the progenitor cells to acquire features characteristic of cancer cells. During this oncogenic transformation, the cells reverted to a more primitive differentiation state and displayed altered histone marks at several key regulatory genes. Science, this issue p. 1529; see also p. 1458

A Phase 2 Trial of Stereotactic Radiosurgery Boost After Surgical Resection for Brain Metastases

PURPOSE To evaluate local control after surgical resection and postoperative stereotactic radiosurgery (SRS) for brain metastases. METHODS AND MATERIALS A total of 49 patients (50 lesions) were enrolled and available for analysis. Eligibility criteria included histologically confirmed malignancy with 1 or 2 intraparenchymal brain metastases, age≥18 years, and Karnofsky performance status (KPS)≥70. A Cox proportional hazard regression model was used to test for significant associations between clinical factors and overall survival (OS). Competing risks regression models, as well as cumulative incidence functions, were fit using the method of Fine and Gray to assess the association between clinical factors and both local failure (LF; recurrence within surgical cavity or SRS target), and regional failure (RF; intracranial metastasis outside of treated volume). RESULTS The median follow-up was 12.0 months (range, 1.0-94.1 months). After surgical resection, 39 patients with 40 lesions were treated a median of 31 days (range, 7-56 days) later with SRS to the surgical bed to a median dose of 1800 cGy (range, 1500-2200 cGy). Of the 50 lesions, 15 (30%) demonstrated LF after surgery. The cumulative LF and RF rates were 22% and 44% at 12 months. Patients who went on to receive SRS had a significantly lower incidence of LF (P=.008). Other factors associated with improved local control include non-small cell lung cancer histology (P=.048), tumor diameter<3 cm (P=.010), and deep parenchymal tumors (P=.036). Large tumors (≥3 cm) with superficial dural/pial involvement showed the highest risk for LF (53.3% at 12 months). Large superficial lesions treated with SRS had a 54.5% LF. Infratentorial lesions were associated with a higher risk of developing RF compared to supratentorial lesions (P<.001). CONCLUSIONS Postoperative SRS is associated with high rates of local control, especially for deep brain metastases<3 cm. Tumors≥3 cm with superficial dural/pial involvement demonstrate the highest risk of LF.