C. Anthony Blau

Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia

CONTEXT The erythropoiesis-stimulating agents (ESAs) erythropoietin and darbepoetin are licensed to treat chemotherapy-associated anemia in patients with nonmyeloid malignancies. Although systematic overviews of trials have identified venous thromboembolism (VTE) risks, none have identified mortality risks with ESAs. OBJECTIVE To evaluate VTE and mortality rates associated with ESA administration for the treatment of anemia among patients with cancer. DATA SOURCES A published overview from the Cochrane Collaboration (search dates: January 1, 1985-April 1, 2005) and MEDLINE and EMBASE databases (key words: clinical trial, erythropoietin, darbepoetin, and oncology), the public Web site of the US Food and Drug Administration and ESA manufacturers, and safety advisories (search dates: April 1, 2005-January 17, 2008). STUDY SELECTION Phase 3 trials comparing ESAs with placebo or standard of care for the treatment of anemia among patients with cancer. DATA EXTRACTION Mortality rates, VTE rates, and 95% confidence intervals (CIs) were extracted by 3 reviewers from 51 clinical trials with 13 611 patients that included survival information and 38 clinical trials with 8172 patients that included information on VTE. DATA SYNTHESIS Patients with cancer who received ESAs had increased VTE risks (334 VTE events among 4610 patients treated with ESA vs 173 VTE events among 3562 control patients; 7.5% vs 4.9%; relative risk, 1.57; 95% CI, 1.31-1.87) and increased mortality risks (hazard ratio, 1.10; 95% CI, 1.01-1.20). CONCLUSIONS Erythropoiesis-stimulating agent administration to patients with cancer is associated with increased risks of VTE and mortality. Our findings, in conjunction with basic science studies on erythropoietin and erythropoietin receptors in solid cancers, raise concern about the safety of ESA administration to patients with cancer.

HIF induces human embryonic stem cell markers in cancer cells.

Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition

The function of metabolic state in stemness is poorly understood. Mouse embryonicstem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent statesrepresenting the inner cell mass (ICM) and epiblast embryos. Human embryonic stemcells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic differencebetween these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalentin their energy production, dynamically switching from glycolysis to mitochondrialrespiration on demand. Despite having a more developed and expanding mitochondrialcontent, EpiSC/hESC have low mitochondrial respiratory capacity due to lowcytochrome c oxidase (COX) expression. Similarly, in vivo epiblastssuppress COX levels. These data reveal EpiSC/hESC functional similarity to theglycolytic phenotype in cancer (Warburg effect). We further show thathypoxia‐inducible factor 1α (HIF1α) is sufficient to drive ESC to aglycolytic Activin/Nodal‐dependent EpiSC‐like stage. This metabolic switch duringearly stem‐cell development may be deterministic.

Derivation of naïve human embryonic stem cells

Significance We report on generation of nontransgenic, naïve human pluripotent cells that represent the developmentally earliest state described for human established cells. Existing human ES cell lines in the later primed state can be toggled in reverse to naïve by exposure to histone deacetylase inhibitors prior to naïve culture. A new line was established directly from an eight-cell embryo under naïve culture conditions. We describe the naïve state in humans and show that naïve human ES cells have expanded endoderm developmental capacity. The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222–9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

American Society of Gene Therapy (ASGT) Ad Hoc Subcommittee on Retroviral-Mediated Gene Transfer to Hematopoietic Stem Cells

Gene transfer using retroviral vectors has been under clinical study for more than 12 years1. Many studies have targeted hematopoietic stem cells (HSCs) as a potentially enduring and renewable source of gene-modified blood cells for the treatment of specific genetic diseases, cancer, leukemia, and HIV-1 infection2. Although initial studies were hampered by very low levels of gene transfer to HSCs, incremental progress has been realized in the efficiency of gene transfer to HSCs. These advances have culminated in the report of clinically significant restoration of immunity in patients with the X-linked form of severe combined immune deficiency (XSCID) by Alain Fischer, Marina Cavazzana-Calvo, and colleagues at the Hopital Necker Enfants Malades in Paris3. Their study and those conducted by Adrian Thrasher and colleagues at the Great Ormond Street Childrens Hospital in London for XSCID and by Claudio Bordignon and colleagues at the Hospital San Raffaele in Milan for children with SCID due to deficiency of adenosine deaminase (ADA) provide incontrovertible proof that gene therapy can ameliorate genetic diseases4,5.

Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells.

Recent evidence indicates that mouse and human embryonic stem cells (ESCs) are fixed at different developmental stages, with the former positioned earlier. We show that a narrow concentration of the naturally occurring short-chain fatty acid, sodium butyrate, supports the extensive self-renewal of mouse and human ESCs, while promoting their convergence toward an intermediate stem cell state. In response to butyrate, human ESCs regress to an earlier developmental stage characterized by a gene expression profile resembling that of mouse ESCs, preventing precocious Xist expression while retaining the ability to form complex teratomas in vivo. Other histone deacetylase inhibitors (HDACi) also support human ESC self-renewal. Our results indicate that HDACi can promote ESC self-renewal across species, and demonstrate that ESCs can toggle between alternative states in response to environmental factors.

Characterization of microRNAs Involved in Embryonic Stem Cell States

Studies of embryonic stem cells (ESCs) reveal that these cell lines can be derived from differing stages of embryonic development. We analyzed common changes in the expression of microRNAs (miRNAs) and mRNAs in 9 different human ESC (hESC) lines during early commitment and further examined the expression of key ESCenriched miRNAs in earlier developmental states in several species. We show that several previously defined hESC-enriched miRNA groups (the miR-302, -17, and -515 families, and the miR-371-373 cluster) and several other hESC-enriched miRNAs are down-regulated rapidly in response to differentiation. We further found that mRNAs up-regulated upon differentiation are enriched in potential target sites for these hESC-enriched miRNAs. Interestingly, we also observed that the expression of ESC-enriched miRNAs bearing identical seed sequences changed dynamically while the cells transitioned through early embryonic states. In human and monkey ESCs, as well as human-induced pluripotent stem cells (iPSCs), the miR-371-373 cluster was consistently up-regulated, while the miR-302 family was mildly down-regulated when the cells were chemically treated to regress to an earlier developmental state. Similarly, miR-302b, but not mmu-miR-295, was expressed at higher levels in murine epiblast stem cells (mEpiSC) as compared with an earlier developmental state, mouse ESCs. These results raise the possibility that the relative expression of related miRNAs might serve as diagnostic indicators in defining the developmental state of embryonic cells and other stem cell lines, such as iPSCs. These data also raise the possibility that miRNAs bearing identical seed sequences could have specific functions during separable stages of early embryonic development.

In vivo selection using a cell-growth switch

A major obstacle to stem-cell gene therapy rests in the inability to deliver a gene into a therapeutically relevant fraction of stem cells. One way to circumvent this obstacle is to use selection. Vectors containing two linked genes serve as the basis for selection, with one gene encoding a selectable product and the other, a therapeutic protein. Applying selection in vivo has the potential to bring a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a ‘cell-growth switch’, allowing a minor population of genetically modified cells to be inducibly amplified, thereby averting the risks associated with cytotoxic drugs. This system provides a general platform for conditionally expanding genetically modified cell populations in vivo, and may have widespread applications in gene and cell therapy.

Granulocytosis causing sickle-cell crisis

mgikg), or topiramate (25 mg/kg) or a combination of the two by intraperitoneal injection. A control group received vehicle (0.9% saline) alone. An hour later, each animal was given 85 mgkg pentylenetetrazol subcutaneously; the time taken to the first generalised seizure was rec~rded .~ A cut-off point of 15 min was set. Although neither topiramate or lamotrigine exerted an effect, none of the animals pre-treated with the combination experienced a seizure throughout the observation period. It is not possible to be certain that the combination (and not topiramate alone) was the effective strategy in these patients. Nevertheless, although lamotrigine was administered to the limit of tolerability, doses of topiramate remained quite small: add-on regulatory trials with topiramate reported maximum seizure reduction with 400-800 mg daily.’ The study in mice also supports particular efficacy for the combination. This may be a consequence of the drugs’ wide range of mechanisms of action affecting Na’ and Ca” channels, and GABA-mediated inhibition and excitatory aminoacid neurotransmission.

Controlled-rate freezing of human ES cells.

A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.