Nuclear reprogramming of human somatic cells by xenopus egg extract requires BRG1

Animal cloning by nuclear transplantation in amphibia was demonstrated almost half a century ago and raised the question of the mechanisms and genes involved in nuclear reprogramming. Here, we demonstrate nuclear reprogramming of permeabilized human cells using extracts from Xenopus laevis eggs and early embryos. We show upregulation of pluripotency markers Oct-4 and germ cell alkaline phosphatase (GCAP) in 293T cells and human primary leukocytes. Reprogrammed leukocytes had a limited life span and did not express surface antigens characteristic of pluripotent cells, indicating that reprogramming was incomplete. Reprogramming activity was detected in egg and early embryo extracts until early blastula stage. Late blastula-stage extracts were not only inactive but also inhibitory to reprogramming. Screening for factors required for reprogramming identified the chromatin remodeling ATPase BRG1. Antibody depletion of BRG1 protein or expression of dominant-negative BRG1 abolished the reprogramming ability of amphibian extracts. Conversely, overexpression of BRG1 in Xenopus animal caps extended their competence from blastula to gastrula stage to respond to basic fibroblast growth factor (bFGF) treatment with induction of the mesodermal marker Xbra. Dissection of the molecular machinery using a simplified assay system may aid in achieving complete nuclear reprogramming of somatic cells for regenerative medicine.     

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1–8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.     

Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells

Functional reprogramming of a differentiated cell toward pluripotency may have long-term applications in regenerative medicine. We report the induction of dedifferentiation, associated with genomewide programming of gene expression and epigenetic reprogramming of an embryonic gene, in epithelial 293T cells treated with an extract of undifferentiated human NCCIT carcinoma cells. 293T cells exposed for 1 h to extract of NCCIT cells, but not of 293T or Jurkat T-cells, form defined colonies that are maintained for at least 23 passages in culture. Microarray and quantitative analyses of gene expression reveal that the transition from a 293T to a pluripotent cell phenotype involves a dynamic up-regulation of hundreds of NCCIT genes, concomitant with down-regulation of 293T genes and of indicators of differentiation such as A-type lamins. Up-regulated genes encompass embryonic and stem cell markers, including OCT4, SOX2, NANOG, and Oct4-responsive genes. OCT4 activation is associated with DNA demethylation in the OCT4 promoter and nuclear targeting of Oct4 protein. In fibroblasts exposed to extract of mouse embryonic stem cells, Oct4 activation is biphasic and RNA-PolII dependent, with the first transient rise of Oct4 up-regulation being necessary for the second, long-term activation of Oct4. Genes characteristic of multilineage differentiation potential are also up-regulated in NCCIT extract-treated cells, suggesting the establishment of "multilineage priming." Retinoic acid triggers Oct4 down-regulation, de novo activation of A-type lamins, and nestin. Furthermore, the cells can be induced to differentiate toward neurogenic, adipogenic, osteogenic, and endothelial lineages. The data provide a proof-of-concept that an extract of undifferentiated carcinoma cells can elicit differentiation plasticity in an otherwise more developmentally restricted cell type.     

Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts

It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone B4 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.     

Hepatitis C virus infection of human T lymphocytes is mediated by CD5

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease. Although infection of hepatocytes is mainly responsible for manifestations of hepatitis C, the virus also invades the immune system by a yet-to-be-identified mechanism. Using human T cell lines and primary T lymphocytes as targets and patient-derived HCV as inocula, we aimed to identify how HCV gains entry into these cells. HCV replication was determined by detection of the HCV RNA replicative (negative) strand and viral proteins, while specific antibodies, knocking down gene expression and making otherwise-resistant cells prone to HCV, were employed to identify a receptor molecule determining T lymphocyte permissiveness to HCV infection. The results revealed that T cell susceptibility to HCV requires CD5, a lymphocyte-specific glycoprotein belonging to the scavenger receptor cysteine-rich family. Blocking of T cell CD5 with antibody or silencing with specific short hairpin RNA (shRNA) decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the opposite effect. Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated infection of these otherwise HCV-resistant cells. In contrast to T cells, hepatocytes do not express CD5. The data revealed that CD5 is a molecule important for HCV entry into human T lymphocytes. This finding provides direct insight into the mechanism of HCV lymphotropism and defines a target for potential interventions against HCV propagating in this extrahepatic compartment.     

Dedifferentiation of cells: new approaches

Reprogramming of a differentiated cell into a cell capable of giving rise to many different cell types, a pluripotent cell, which in turn could repopulate or repair sick or damaged tissue, would present beneficial applications in regenerative medicine. Somatic cell nuclear transfer may offer this possibility, but technical hurdles and ethical frameworks currently prevent application of this technology in several countries. As a result, alternative strategies to reprogramming cell fate are being developed. This review briefly addresses somatic cell nuclear transfer and focuses on recent non-nuclear transfer-based approaches for reprogramming somatic cells and enhancing their differentiation potential. These include the fusion of somatic cells with embryonic stem cells, the treatment of somatic cells with extract of pluripotent cells and the retroviral transduction of somatic cells to overexpress pluripotency genes.     

Aberrant expression of the insulin-like growth factor-1 receptor by T cells from patients with Graves' disease may carry functional consequences for disease pathogenesis

Graves' disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like growth factor receptor-1 (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD. IGF-1R on fibroblasts, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 +/- 4% (mean +/- SE; n = 33) in patients with GD compared with 15 +/- 3% (n = 21; p < 10(-8)) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and IGF-1R+CD4+CD3+ cells as those found in the peripheral circulation. GD-derived peripheral T cells express durable, constitutive IGF-1R expression in culture and receptor levels are further up-regulated following CD3 complex activation. IGF-1 enhanced GD-derived T cell incorporation of BrdU (p < 0.02) and inhibited Fas-mediated apoptosis (p < 0.02). These findings suggest a potential role for IGF-1R displayed by lymphocytes in supporting the expansion of memory T cells in GD.     

Function of a heterologous muscarinic receptor in T cell antigen receptor signal transduction mutants

Previously we have described a system of somatic cell genetics (J.CaM1 and J.CaM2) for analyzing signal transduction via the T cell antigen receptor complex (CD3/Ti). Here we describe a third mutant, J.CaM3, which also expresses high levels of receptors that are functionally impaired. Like J.CaM1, J.CaM3 demonstrates partial signal transduction via CD3/Ti to only certain stimuli. J.CaM1, J.CaM2, and J.CaM3 define three non-Ti complementation groups involved in receptor function. To evaluate the mutations further we have introduced a heterologous receptor, the human muscarinic receptor 1 (HM1), into the parental Jurkat and mutant cell lines. This receptor demonstrates signal transduction competence in all these hosts, indicating that 1) T cells express the necessary apparatus for the coupling of HM1 to second messenger generation and 2) the mutations in the J.CaM family all affect molecules that are specific to CD3/Ti, and not HM1, function. Finally, the HM1 receptor exhibits partial sensitivity to cholera toxin in Jurkat cells, in contrast to the virtually complete sensitivity of CD3/Ti to cholera toxin.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming

The bioenergetics of somatic dedifferentiation into induced pluripotent stem cells remains largely unknown. Here, stemness factor-mediated nuclear reprogramming reverted mitochondrial networks into cristae-poor structures. Metabolomic footprinting and fingerprinting distinguished derived pluripotent progeny from parental fibroblasts according to elevated glucose utilization and production of glycolytic end products. Temporal sampling demonstrated glycolytic gene potentiation prior to induction of pluripotent markers. Functional metamorphosis of somatic oxidative phosphorylation into acquired pluripotent glycolytic metabolism conformed to an embryonic-like archetype. Stimulation of glycolysis promoted, while blockade of glycolytic enzyme activity blunted, reprogramming efficiency. Metaboproteomics resolved upregulated glycolytic enzymes and downregulated electron transport chain complex I subunits underlying cell fate determination. Thus, the energetic infrastructure of somatic cells transitions into a required glycolytic metabotype to fuel induction of pluripotency.     

Non-Genetic Direct Reprogramming and Biomimetic Platforms in a Preliminary Study for Adipose-Derived Stem Cells into Corneal Endothelia-Like Cells

Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.     

A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor

A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation.