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Since the discovery of induced pluripotent stem cells (iPSCs),numerous approaches have been explored to improve the original protocol,which is based on a two-dimensional (2D) cell-culture system. Surprisingly,nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here,we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness,degradability and biochemical composition,we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity. View Publication

The developmental potential of human ES cells makes them an important tool in developmental,pharmacological,and clinical research. For human ES cell technology to be fully exploited,however,culture efficiency must be improved,large-scale culture enabled,and safety ensured. Traditional human ES cell culture systems have relied on serum products and mouse feeder layers,which limit the scale,present biological variability,and expose the cells to potential contaminants. Defined,feeder-independent culture systems improve the safety and efficiency of ES cell technology,enabling translational research. The protocols herein are designed with the standard research laboratory in mind. They contain recipes for the formulation of mTeSR (a defined medium for human ES cell culture) and detailed protocols for the culture,transfer,and passage of cells grown in these feeder-independent conditions. They provide a basis for routine feeder-independent culture,and a starting point for additional optimization of culture conditions. View Publication

Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™),which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives,expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for textgreater 20 passages on tissue culture-treated polystyrene plates,coated from 5 μg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-,endo-,and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy,atomic force microscopy,and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density,via the concentration of depositing solution,revealed a threshold surface density of 250 ng/cm²,which is required for hESCs attachment,proliferation,and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable. View Publication

BACKGROUND: Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells,such as embryonic stem cells and induced pluripotent stem cells,appear to have a hypermethylated status compared with differentiated cells. However,the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally,differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.backslashnbackslashnMETHODOLOGY: Here,we determined the DNA methylation profiles of 10 human cell lines,including 2 ESC lines,4 virally derived iPSC lines,2 episomally derived iPSC lines,and the 2 parental cell lines from which the iPSCs were derived using Illumina's Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness,whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.backslashnbackslashnCONCLUSIONS: This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods,the corresponding somatic cells,and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine. View Publication

Hypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2??-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined,these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here,we show that low O2 tension and hypoxiainducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies,we implicate O2 tension as a modifier of a key cell fate decision,namely whether neural progenitors differentiate toward neurons or glia. Furthermore,our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC,a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage.We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types. View Publication

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential,previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage,culture milieu,and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin(-) BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA,39% and LS,28%). A second sequential enrichment for the dihydropyridine receptor subunit alpha2delta1 (DHPR-alpha2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1(+) enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca(2+) transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion,IP CD117/Sca-1(+) murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore,temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-alpha2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation. View Publication

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted,but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here,we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly,loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly,null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation,thereby accelerating hematopoietic regeneration. Consistent with these findings,Puma is required for radiation-induced apoptosis in HSCs and HPCs,and Puma is selectively induced by irradiation in primitive hematopoietic cells,and this induction is impaired in Puma-heterozygous cells. Together,our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy. View Publication

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However� View Publication

Tristetraprolin (TTP,Zfp36,Nup475,Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements in their 3' untranslated regions. Through this mechanism,TTP functions as a rheostatic,temporal regulator of gene expression. TTP knockout (KO) mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell compartment in TTP KO mice is also altered. Although no change was detected in long-term hematopoietic stem cell (HSC) frequency or function,as assayed by immunophenotypic markers or limiting dilution transplants,we observed increases in the frequencies and numbers of short-term HSCs,multipotent progenitors,and granulocyte-monocyte progenitors. This pattern is consistent with reactive granulopoiesis� View Publication

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) $$$$ and $$$$ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. View Publication

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. View Publication

Protein delivery allows a clinical effect to be directly realized without genetic modification of the host cells. We have developed a cationic bolaamphiphile as a non-viral vector for protein delivery application. The relatively low toxicity and efficient protein delivery by the cationic bolaamphiphile prompted us to test the system for the generation of induced pluripotent stem cells (iPSCs) as an alternative to the conventional vector-based genetic approach. Studies on the kinetics and cytotoxicity of the protein delivery system led us to use an optimized cationic bolaamphiphile-protein complex ratio of 7:1 (wt/wt) and a 3 h period of incubation with human fibroblasts,to ensure complete and non-toxic protein delivery of the reprogramming proteins. The reprogrammed cells were shown to exhibit the characteristics of embryonic stem cells,including expression of pluripotent markers,teratoma formation in SCID mice,and ability to be differentiated into a specific lineage,as exemplified by neuronal differentiation. View Publication