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Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation. (Blood. 2011;117(19):5057-5066) IntroductionHematopoietic stem cells (HSCs) can self-renew and differentiate into all cell types of the hematopoietic system and are regulated by interacting intrinsic and extrinsic mechanisms. 1 Among intrinsic mechanisms, several transcriptional regulators involved as partners of leukemogenic fusion proteins, such as Mll 2-4 and Evi1, 5 are required for normal HSC function, whereas others, such as Runx1 6,7 and Scl,8,9 are essential for the establishment of HSCs during development. PR domain-containing 16 (PRDM16), a 140-kDa zinc finger protein, was originally discovered as a fusion partner in t(1:3)(p36;q21) translocations in acute myeloblastic leukemia (AML) 10,11 and later in t(1;21)(p36;q22) translocations fused to RUNX1. 12,13 In addition, elevated PRDM16 expression, because of promoter hypomethylation, is frequently observed in karyotypically normal AML. 14 Deletion of the PR domain, which shows homology with a SET chromatin remodeling domain and is also present in EVI1, 10 appears important for the leukemogenic properties of human PRDM16. Translocations involving PRDM16 invariably delete the PR domain, 10-13 whereas PR-deleted Prdm16 causes AML in p53 Ϫ/Ϫ mice. 14 Furthermore, both Prdm16 and Evi1 are frequent targets of insertional mutagenesis in mice, causing deletion of the PR domain. 15 Overexpression of Prdm16 expands HSCs in vitro. However, these expanded HSCs cause a myeloproliferative disease after transplantation. 16 Prdm16 has also been shown to be critical for the development of brown adipose tissue in the mouse. PRDM16 is a transcriptional cofactor and interacts with the ligand-activated transcription factor peroxisome proliferatoractivated receptor-␥ and with CCAAT/enhancer-binding protein-␤. 17,18 Although its involvement in leukemic transloc...

We develop biodegradable polymeric nanoparticles to facilitate non-viral gene transfer to human embryonic stem cells (hESC). Small (~200 nm) and positively charged (~10 mV) particles are formed by the self-assembly of cationic, hydrolytically-degradable, poly(beta-amino esters) and plasmid DNA. Varying the end-group of the polymer can tune the biophysical properties of the resulting nanoparticles and their gene delivery efficacy. An OCT4 driven GFP hES cell line was created to allow rapid identification of nanoparticles that facilitate gene transfer while maintaining an hESC undifferentiated state. Using this cell system, we synthesized nanoparticles that have gene delivery efficacy up to four times higher than the leading commercially available transfection agent, Lipofectamine 2000. Importantly, these materials have minimal toxicity and do not adversely affect hES colony morphology or cause non-specific differentiation. KeywordsGene Delivery; Nanoparticle; Stem Cell; Transfection; Polymer The delivery of genes to stem cells can advance cell-based therapies and the field of tissue engineering. Given the right conditions, pluripotent stem cells can potentially differentiate into any cell type of the body, allowing wide therapeutic utility including treatments for autoimmune diseases, spinal chord injury, Parkinson's disease, and cardiac tissue engineering, among many others. 1 Gene delivery could allow for directed differentiation from a pluripotent stem cell into specific differentiated cell types of interest including hematopoietic cells, neurons, cardiomycytes, and osteoblasts, as well as reprogramming a differentiated cell back into a pluripotent state. 2 -4 Beyond controlled differentiation, ectopic expression of key growth and transcription factors could allow for elucidation of fundamental cell development pathways in vitro as well as regulation of growth in vivo once the cells are transferred to a patient. Gene delivery can also provide a mechanism for in vivo expression of secreted therapeutic proteins. We have developed a class of polymers, poly ( -amino esters), that are promising for nonviral gene delivery due to their ability to condense DNA into nanoparticles that facilitate cellular uptake and endosomal escape. 10 -12 Due to hydrolytic cleavage of the ester groups that compose these polymers, they are biodegradable and have low toxicity. 10 , 13 , 14 These particles are also useful as they can be coated for ligand-specific delivery. 15 Chemical modification of the ends of these polymers results in improved biomaterials that can deliver DNA to human umbilical vein endothelial cells (HUVECs) at levels comparable to adenovirus. 16 , 17 Here we develop polymeric nanoparticles for non-viral gene transfer to undifferentiated hESCs. To ensure that the transfected hESC colonies remain in an undifferentiated state once transfected, we use hESCs targeted to stably express Oct4-driven GFP and monitor GFP levels as an indicator of the undifferentiated state of the hESCs. We demonstrate that these nano...

We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.