scholarly journals Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency

2012 ◽  
Vol 197 (6) ◽  
pp. 731-745 ◽  
Author(s):  
Jian Qu ◽  
J. Michael Bishop
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem ◽  
Morphological Differentiation ◽  
Ips Cells ◽  
Mammary Epithelial ◽  
Rapid Transit ◽  
Self Renewal ◽  
Induced Pluripotent ◽  
Cell Cycle Inhibitors

Nucleostemin (NS) is a nucleolar GTP-binding protein that was first identified in neural stem cells, the functions of which remain poorly understood. Here, we report that NS is required for mouse embryogenesis to reach blastulation, maintenance of embryonic stem cell (ESC) self-renewal, and mammary epithelial cell (MEC) reprogramming to induced pluripotent stem (iPS) cells. Ectopic NS also cooperates with OCT4 and SOX2 to reprogram MECs and mouse embryonic fibroblasts to iPS cells. NS promotes ESC self-renewal by sustaining rapid transit through the G1 phase of the cell cycle. Depletion of NS in ESCs retards transit through G1 and induces gene expression changes and morphological differentiation through a mechanism that involves the MEK/ERK protein kinases and that is active only during a protracted G1. Suppression of cell cycle inhibitors mitigates these effects. Our results implicate NS in the maintenance of ESC self-renewal, demonstrate the importance of rapid transit through G1 for this process, and expand the known classes of reprogramming factors.

308 Tet1 PLAYS IMPORTANT ROLES IN MAINTAINING CHARACTERIZATIONS OF PORCINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 301
Author(s):  
A. R. Fan ◽  
K. Y. Ma ◽  
T. C. Zhao ◽  
P. P. An ◽  
B. Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Qrt Pcr ◽  
Post Transfection ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

scholarly journals Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases, including Muscular Dystrophies

2020 ◽  
Vol 21 (15) ◽  
pp. 5467
Author(s):  
Daniela Gois Beghini ◽  
Samuel Iwao Horita ◽  
Cynthia Machado Cascabulho ◽  
Luiz Anastácio Alves ◽  
Andrea Henriques-Pons
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Ips Cells ◽  
High Plasticity ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krüppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

Human induced pluripotent stem cells are capable of B-cell lymphopoiesis

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. 4008-4011 ◽  
Author(s):  
Lee Carpenter ◽  
Ram Malladi ◽  
Cheng-Tao Yang ◽  
Anna French ◽  
Katherine J. Pilkington ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cell ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Ips Cell ◽  
Lymphoid Lineage ◽  
Human Ips Cells ◽  
Induced Pluripotent ◽  
First Time ◽  
Unique Potential

Abstract Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45+CD19+CD10+ and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45+CD19+ cells also exhibited multiple genomic D-JH rearrangements, which supports a pre–B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.

scholarly journals Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Yinxiang Wang ◽  
Jessica Aijia Liu ◽  
Keith K. H. Leung ◽  
Mai Har Sham ◽  
Danny Chan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Early Stage ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Ips Cells ◽  
Egfp Expression ◽  
Retroviral Transduction ◽  
Intramembranous Bone ◽  
Induced Pluripotent

Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

305 TRANSPOSON-MEDIATED REPROGRAMMING OF LIVESTOCK SOMATIC CELLS TO INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 300
Author(s):  
T. R. Talluri ◽  
D. Hermann ◽  
B. Barg-Kues ◽  
K. Debowski ◽  
R. Behr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Insertional Mutagenesis ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Sleeping Beauty ◽  
Major Step ◽  
Increased Risk ◽  
Induced Pluripotent

The elusive nature of embryonic stem cells in livestock makes reprogramming of somatic cells to induced pluripotent stem (iPS) cells a promising approach for targeted genetic modifications. The first attempts to produce iPS cells from livestock species were made using retro- and lentiviral vectors, which are associated with an increased risk of insertional mutagenesis and which are not easily removable after reprogramming. Here, we describe a nonviral method for the derivation of porcine and bovine iPS cells, using Sleeping Beauty (SB) and piggyBac (PB) transposon systems. The transposons encode the murine or primate reprogramming factors OCT4, SOX2, KLF4, MYC, and LIN28, separated by self-cleaving peptide sequences, respectively. In addition, the PB transposon cassette contains a NANOG-cDNA. The SB or PB transposon-reprogrammed porcine iPS cells expressed typical markers of embryonic stem cells (SSEA1, SSEA4, TRA-1-60, and endogenous stemness genes), showed long-term proliferation under feeder-free culture conditions, differentiated into cell types of the 3 germ layers in vitro, and formed teratomas after subcutaneous injection into immune-deficient nude mice. Both transposon systems are currently being tested in bovine fibroblasts. The results are a major step towards the derivation of authentic porcine and bovine iPS cells, in which the transposon transgenes can be eliminated after reprogramming.

scholarly journals Differentiation of Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells into Steroid-Producing Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (9) ◽  
pp. 4336-4345 ◽  
Author(s):  
Takuhiro Sonoyama ◽  
Masakatsu Sone ◽  
Kyoko Honda ◽  
Daisuke Taura ◽  
Katsutoshi Kojima ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regulatory Protein ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroidogenic Factor 1 ◽  
Differentiated Cells ◽  
Steroidogenic Acute Regulatory ◽  
Induced Pluripotent ◽  
Mesodermal Lineage

Although there have been reports of the differentiation of mesenchymal stem cells and mouse embryonic stem (ES) cells into steroid-producing cells, the differentiation of human ES/induced pluripotent stem (iPS) cells into steroid-producing cells has not been reported. The purpose of our present study was to establish a method for inducing differentiation of human ES/iPS cells into steroid-producing cells. The first approach we tried was embryoid body formation and further culture on adherent plates. The resultant differentiated cells expressed mRNA encoding the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, CYP17A1, and CYP19, and secreted progesterone was detected in the cell medium. However, expression of human chorionic gonadotropin was also detected, suggesting the differentiated cells were trophoblast like. We next tried a multistep approach. As a first step, human ES/iPS cells were induced to differentiate into the mesodermal lineage. After 7 d of differentiation induced by 6-bromoindirubin-3′-oxime (a glycogen synthase kinase-3β inhibitor), the human ES/iPS cells had differentiated into fetal liver kinase-1- and platelet derived growth factor receptor-α-expressing mesodermal lineage cells. As a second step, plasmid DNA encoding steroidogenic factor-1, a master regulator of steroidogenesis, was introduced into these mesodermal cells. The forced expression of steroidogenic factor-1 and subsequent addition of 8-bromoadenosine 3′,5′-cyclic monophosphate induced the mesodermal cells to differentiate into the steroidogenic cell lineage, and expression of CYP21A2 and CYP11B1, in addition to steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11A1, and CYP17A1, was detected. Moreover, secreted cortisol was detected in the medium, but human chorionic gonadotropin was not. These findings indicate that the steroid-producing cells obtained through the described multistep method are not trophoblast like; instead, they exhibit characteristics of adrenal cortical cells.

scholarly journals EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6314 ◽  
Author(s):  
Miaoying Yu ◽  
Yinghui Wei ◽  
Kui Xu ◽  
Shasha Liu ◽  
Lei Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Epigenetic Modification ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Specific Inhibitor ◽  
Nuclear Antigen ◽  
Self Renewal ◽  
Egfr Inhibition

Background Self-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood. Methods Here, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478. Results EGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G0/G1 phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs. Conclusions Taken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.

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