搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless,a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction of hepatocyte maturation,oxidative phosphorylation is essential at all stages of differentiation. View Publication

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands,targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features,and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall,the approach described here allows for the rapid generation of human cardiac grafts from hPSCs,in a total of 24 days,providing a suitable platform for cardiac engineering and disease modeling in the human setting. View Publication

MicroRNAs (miRNA) comprise a group of short ribonucleic acid molecules implicated in regulation of key biological processes and functions at the post-transcriptional level. Ionizing radiation (IR) causes DNA damage and generally triggers cellular stress response. However,the role of miRNAs in IR-induced response in human embryonic stem cells (hESC) has not been defined yet. Here,by using system biology approaches,we show for the first time,that miRNAome undergoes global alterations in hESC (H1 and H9 lines) after IR. Interrogation of expression levels of 1,090 miRNA species in irradiated hESC showed statistically significant changes in 54 genes following 1 Gy of X-ray exposures; global miRNAome alterations were found to be highly temporally and cell line--dependent in hESC. Time-course studies showed that the 16 hr miRNAome radiation response of hESC is much more robust compared to 2 hr-response signature (only eight genes),and may be involved in regulating the cell cycle. Quantitative real-time PCR performed on some miRNA species confirms the robustness of our miRNA microarray platform. Positive regulation of differentiation-,cell cycle-,ion transport- and endomembrane system-related processes were predicted to be negatively affected by miRNAome changes in irradiated hESC. Our findings reveal a fundamental role of miRNAome in modulating the radiation response,and identify novel molecular targets of radiation in hESC. View Publication

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However,no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work,we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF,EGF and PDGF. These cells are SSEA4(+),OCT3/4(+),NANOG(+),SOX2(+),LIN28(+),CD13(+),CD105(+),CD34(-),CD45(-),CD90(+),CD29(+),CD73(+),STRO1(+) and CD146(-),and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly,DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm,endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4,GATA6,MIXL1,NANOG,OCT3/4,SOX1 and SOX2 to determine the degree of similarity between DPPSCs,EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs,hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages,they represent an easily accessible source of stem cells,which opens a range of new possibilities for regenerative medicine. View Publication

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development,but the soundness of this model has been challenged by others,who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h,with minimal expression of mesoderm markers,including T (Brachyury). Instead,they begin to express a series of trophoblast markers,including HLA-G,demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium,and,over time,produce extensive amounts of human chorionic gonadotropin,progesterone,placental growth factor,and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling,which at day 2 provide colonies that are entirely KRT7+ and in which the majority of cells are transiently CDX2+. Colonies grown on two chemically defined media,including the one in which BMP4 was reported to drive mesoderm formation,also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts. View Publication

BACKGROUND: The dose-limiting toxicity of doxorubicin on hematopoietic stem cells reduces the maximum benefit from this powerful drug. Melatonin may play a role in reducing this toxicity. MATERIALS AND METHODS: Melatonin at 10 microM was used while challenging human peripheral blood stem cells (PBSC) with doxorubicin (0.6 microM and 1 microM),and colony formation was used to evaluate the protective effect of melatonin. RESULTS: Melatonin was protective for the myeloid and erythroid series when given during or 1 hour after,but not before,doxorubicin,as measured by colony assay. This protection was independent from its antioxidant function as measured by 2',7'-dichlodihydro-fluorescein diacetate and was selective for PBSC when compared to the MCF-7 cancer cell line. CONCLUSION: The results suggest the importance of the time sequence for melatonin administration to exert its protective effect in relation to doxorubicin treatment,as well as its protective effect on both erythroid and myeloid elements independent from its antioxidant function. View Publication

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover,safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple,nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe,efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research,disease modeling,and regenerative medicine. ?? 2010 Elsevier Inc. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction,as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick((TM))E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6),or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml,respectively,and nucleated cell (NC) content of 9.7 and 23.8 x 10(8) resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%),CD34(+) cells (76.3%-79.0%),and colony-forming cells (64.1%-86.3%). Moreover,we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast,the mean depletion rate using BC processing proved to be significantly different for PLTs (10%,p = 0.03) and RBCs (39.6%,p textless 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p textless 0.01),compared with a less significant MNC increase in the BC group (p textless 0.05). For mice transplanted with WB-derived progenitors,we observed a mean of 15.3% +/- 15.5% of human CD45(+) cells within the BM compared with 19.9% +/- 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34(+) cells was 4.2% +/- 3.1% for WB samples and 4.5% +/- 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration,no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuick((TM))E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant,including a partial depletion of granulocytes,RBCs,and PLTs without the need for centrifugation. Therefore,it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans. View Publication

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies,but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice,whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases,the regenerated cells showed the same clonal markers (trisomy 8,n = 3; and 5q-,n = 1) as the original sample and,in one instance,regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly,the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3,granulocyte-macrophage colony-stimulating factor,and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS. View Publication

Our ability to guide differentiation of human pluripotent stem cells (hPSCs) toward desired lineages efficiently and reproducibly in xeno-free conditions is the key to advancing hPSC technology from the laboratory to clinical use. Here we report an engineered biomimetic substrate functionalized with both peptide ligands for α5β1 and α6β1 integrins to support efficient early mesodermal differentiation of human embryonic stem cells (hESCs) when cultured in a differentiation medium containing BMP4. In contrast,mesodermal differentiation is not induced on substrates functionalized with either ligand alone even though the culture medium is identical. Mesodermal differentiation was characterized by immunofluorescent staining,flow cytometric analysis,and RT-PCR analysis of early mesodermal markers Brachyury,Mixl1,and Wnt3. The early mesodermal progenitors derived on the substrate functionalized with both integrin ligands have the normal developmental potential to further differentiate along the hemato-endothelial and cardiac lineages. Immobilized ligands for α5β1 and α6β1 integrins both are permissive,necessary,and sufficient insoluble ligands in this engineered system to support early mesodermal differentiation of hESCs. This synthetic substrate,in conjunction with defined soluble factors,constructs a well-controlled and xeno-free early mesodermal differentiation niche that offers advantages over the previously reported niche constructed with the Matrigel-coated substrate. View Publication