Ruth M. Risueño

Direct conversion of human fibroblasts to multilineage blood progenitors

As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.

Identification of Drugs Including a Dopamine Receptor Antagonist that Selectively Target Cancer Stem Cells

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

Signal control of hematopoietic stem cell fate: Wnt, Notch, and Hedgehog as the usual suspects.

Purpose of reviewHematopoietic homeostasis depends on appropriate self-renewal and differentiation capacity of hematopoietic stem cells. The characterization of the key extracellular signals that integrate with intracellular molecular machinery to regulate hematopoietic stem cells fate choice is crucial to move toward hematopoietic stem cell clinical application. Recent findingsSeveral factors have been described as positive and negative regulators of hematopoietic stem cell self-renewal and differentiation. Most of the hematopoietic cytokines studied promote either survival or differentiation or both in hematopoietic stem cells ex vivo, whereas morphogens (Wnt, Notch, and Hedgehog) may signify a class of hematopoietic stem cell regulators that support expansion of the hematopoietic stem cell pool by a combination of survival and induced self-renewal. SummaryAlthough Wnt, Notch, and Hedgehog signaling pathways have been implicated in self-renewal and proliferation in vivo, modulation of these pathways alone does not result in substantive expansion of hematopoietic stem cells ex vivo. In addition to these signaling pathways, Bcl-2 family members may have an important role in inducing survival in hematopoietic stem cells both in vivo and ex vivo. Understanding the complex relationship between these unique signaling pathways is essential to achieve successful ex-vivo expansion toward enhanced hematopoietic stem cell transplantation-based therapies.

Inability of Human Induced Pluripotent Stem Cell‐Hematopoietic Derivatives to Downregulate MicroRNAs In Vivo Reveals a Block in Xenograft Hematopoietic Regeneration

Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here we show that, unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC‐derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC‐derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell‐derived hematopoietic cells for transplantation compared with hESCs. Since use of the human–mouse xenograft models prevents detection of putative hiPSC‐derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources. STEM CELLS 2012; 30:131–139.

Identification of T-lymphocytic leukemia–initiating stem cells residing in a small subset of patients with acute myeloid leukemic disease

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs, substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples, 39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However, 33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients, but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction, and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function, and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies.

High levels of global DNA methylation are an independent adverse prognostic factor in a series of 90 patients with de novo myelodysplastic syndrome

The prognostic impact of global DNA methylation and hydroxymethylation was assessed in 90 patients with de novo myelodysplastic syndrome (MDS). DNA was isolated from bone marrow samples obtained at diagnosis and global methylation and hydroxymethylation were determined by ELISA. Patients with a percentage of methylated DNA above 2.73% had a shorter overall survival than those with lower levels (P=0.018) and presented a negative trend in terms of leukemia-free survival (P=0.084), that was statistically significant after censoring 9 patients that received disease-modifying treatments both in univariate and multivariate analyses. Similarly, the low-risk MDS patients defined by the IPSS, WPSS and IPSS-R with 5-mC percentage in total DNA above 2.73% had a shorter overall survival (P=0.032; P=0.023; P=0.031). No cut-off value for the 5-hydroxymethylcytosine percentage with statistical significance for overall or leukemia-free survival was obtained. This study suggests that global DNA methylation predicts overall survival in myelodysplastic syndromes.

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self‐renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98‐HOXA10hd (NA10) on adult murine bone marrow HSC self‐renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45− GlycophorinA+ cells and erythroid progenitors (blast‐forming unit‐erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells. STEM Cells 2011;29:736–741

Sam68 Allows Selective Targeting of Human Cancer Stem Cells

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Emetine induces chemosensitivity and reduces clonogenicity of acute myeloid leukemia cells

Acute myeloid leukemia (AML) is an hematologic neoplasia characterized by the accumulation of transformed immature myeloid cells in bone marrow. Although the response rate to induction therapy is high, survival rate 5-year after diagnosis is still low, highlighting the necessity of new novel agents. To identify agents with the capability to abolish the self-renewal capacity of AML blasts, an in silico screening was performed to search for small molecules that induce terminal differentiation. Emetine, a hit compound, was validated for its anti-leukemic effect in vitro, ex vivo and in vivo. Emetine, a second-line anti-protozoa drug, differentially reduced cell viability and clonogenic capacity of AML primary patient samples, sparing healthy blood cells. Emetine treatment markedly reduced AML burden in bone marrow of xenotransplanted mice and decreased self-renewal capacity of the remaining engrafted AML cells. Emetine also synergized with commonly used chemotherapeutic agents such as ara-C. At a molecular level, emetine treatment was followed by a reduction in HIF-1α protein levels. This study validated the anti-leukemiceffect of emetine in AML cell lines, a group of diverse AML primary samples, and in a human AML-transplanted murine model, sparing healthy blood cells. The selective anti-leukemic effect of emetine together with the safety of the dose range required to exert this effect support the development of this agent in clinical practice.

Targeting LSCs: powering an old tool.

In this issue of Blood , Hassane and colleagues describe the discovery of new therapeutic agents effective against leukemic initiating cells through an in silico approach, demonstrating the power of collective knowledge deposited in publicly available gene-expression databases. Putative leukemic