Prospects for Stem Cell-Based Therapy

Resident pools of somatic stem cells in many organs are responsible for tissue maintenance and repair. The goal of regenerative medicine is to exploit these cells either by transplanting them from an exogenous source or by activating endogenous stem cells pharmacologically. For diseases caused by mutations in a single gene, the therapeutic goal is tissue replacement using stem cells engineered to correct the genetic defect. However, a number of technical hurdles must be overcome before therapies based on pluripotent human stem cells can enter the clinic

Reprogramming Cellular Identity for Regenerative Medicine

Although development leads unidirectionally toward more restricted cell fates, recent work in cellular reprogramming has proven that one cellular identity can strikingly convert into another, promising countless applications in biomedical research and paving the way for modeling diseases with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. Here, we review evidence demonstrating that, because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. We also discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration

microRNAs become macro players in somatic cell reprogramming

Embryonic stem cell specific microRNAs (miRNAs) have previously been shown to enhance the efficiency of transcription-factor-based reprogramming. However, whether reprogramming could be achieved entirely by miRNAs remained unclear. A recent report shows that the expression of the miR-302/367 cluster of miRNAs can directly reprogram somatic cells without the use of any transcription factors. This new method raises interesting questions about the mechanisms of reprogramming and is likely to facilitate the generation of induced pluripotent stem cells for potential future clinical use

Cellular Therapy for Fanconi Anemia: The Past, Present, and Future

Allogeneic hematopoietic cell transplantation (HCT) remains the only proven curative therapy for the hematologic manifestation of Fanconi anemia (FA). Over the past 2 decades, major advances have been made such that transplant outcomes have markedly improved. With the development of in vitro fertilization and preimplantation genetic diagnosis, HLA-matched sibling donor umbilical blood transplantation may be an option for more patients with FA. Recently, the use of pluripotent stem cells has been explored as a novel approach to model the hematopoietic developmental defects in FA, and to provide a potential source of autologous stem cells that can be genetically manipulated and used to generate corrected hematopoietic progenitors

Activation of Phosphatidylinositol 3-Kinase in Cells Expressing abl Oncogene Variants

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants

microRNA Expression during Trophectoderm Specification

Segregation of the trophectoderm from the inner cell mass of the embryo represents the first cell-fate decision of mammalian development. Transcription factors essential for specifying trophectoderm have been identified, but the role of microRNAs (miRNAs) in modulating this fate-choice has been largely unexplored. We have compared miRNA expression in embryonic stem cell (ESC)-derived trophectoderm and in staged murine embryos to identify a set of candidate miRNAs likely to be involved in trophectoderm specification.We profiled embryonic stem cells (ESCs) as they were induced to differentiate into trophectodermal cells by ectopic expression of HRas/Q61L. We also profiled murine embryos at progressive stages of preimplantation development (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst), which includes the time window in which the trophectoderm is specified in vivo Q61L/H.We describe miRNA expression changes that occur during trophectoderm specification and validate that our in vitro system faithfully recapitulates trophectoderm specification in vivo. By comparing our in vitro and in vivo datasets, we have identified a minimal set of candidate miRNAs likely to play a role in trophectoderm specification. These miRNAs are predicted to regulate a host of development-associated target genes, and many of these miRNAs have previously reported roles in development and differentiation. Additionally, we highlight a number of miRNAs whose tight developmental regulation may reflect a functional role in other stages of embryogenesis. Our embryo profiling data may be useful to investigators studying trophectoderm specification and other stages of preimplantation development

A screen to identify drug resistant variants to target-directed anti-cancer agents

The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia (CML)-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair