Abstract 11791: Blockade of Senescence-associated Microrna-195 in Aged Skeletal Myoblasts Facilitates Reprogramming to Produce Induced Pluripotent Stem Cells

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Hideyuki Kondo ◽  
Ha Won Kim ◽  
Lei Wang ◽  
Motoi Okada ◽  
Ronald W Millard ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Autologous Transplantation ◽  
Luciferase Assay ◽  
Rt Pcr ◽  
Skeletal Myoblasts ◽  
Reprogramming Efficiency ◽  
Induced Pluripotent ◽  
Autologous Cell Transplantation

Background: Older age is the major risk factor for heart failure, and reprogramming a patient’s own cells to produce induced pluripotent stem cells (iPSCs) is a promising strategy for autologous cell transplantation therapy. However, low reprogramming efficiency of senescent cells remains as a major pitfall. Recently, our preliminary data suggested that inhibiting senescence-associated miR-195 rejuvenated aged stem cells by reactivating anti-aging defense system. This study investigated the effects of blocking miR-195 expression on the reprogramming efficiency of old skeletal myoblasts (OSkMs). Methods and Results: MiR-195 expression was significantly higher in OSkMs isolated from aged mice (24 months) as compared to those from young mice (2 months), as examined by RT-PCR. OSkMs showed impaired expression of anti-aging factors (Tert and Sirt1) and higher expression of pro-aging markers (p53, p21, p16). Intriguingly, blocking miR-195 expression in OSkMs by transfection with anti-miR-195 significantly induced restoration of Tert and Sirt1 as well as telomere re-lengthening as examined by RT-PCR and quantitative fluorescent in situ hybridization. Luciferase assay confirmed that Sirt1 is one of the direct targets of miR-195 relevant to senescence of OSkMs. Importantly, lower reprogramming efficiency of OSkMs was significantly improved by miR-195 abrogation without altering karyotype or expression of pluripotency markers. Furthermore, iPSCs lacking miR-195 successfully differentiated into all three germ layers, indicating that deletion of miR-195 does not affect pluripotency. Notably, contraction rates were markedly higher in beating cells transfected with anti-miR-195 as compared to that with scramble (68.5±5.6 vs 47.3±2.8/min). Conclusions: Blocking age-induced miR-195 is a novel promising approach for efficient iPSCs generation from senescent cells, which has beneficial for autologous transplantation of iPSCs in elderly patients.

scholarly journals Proteins Reprogramming: Present and Future

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Yang Yang ◽  
Bin Liu ◽  
Jianwen Dong ◽  
Liangming Zhang ◽  
Mao Pang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Chemical Compounds ◽  
Insertion Mutation ◽  
Potential Safety ◽  
Reprogramming Efficiency ◽  
Immunological Rejection ◽  
Clinical Interest ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are of great clinical interest for they are derived from one’s own somatic cells and have the potential of committed differentiation without immunological rejection after autografting. However, the use of viral and other modified vectors may still cause tumorigenesis due to chromosome insertion mutation, leading to limited practical use. iPSCs generated by reprogramming proteins overcome the potential safety risk and complicated manipulation procedures, thus they own better application prospective, yet some technical difficulties need to be studied and resolved, for instance, low reprogramming efficiency, unclear transduction, and reprogramming mechanism. In this paper, we summarize the current progress of proteins reprogramming technology for generation of iPSCs and discuss the promising efficiency-improved reprogramming methods by proteins plus other kinds of chemical compounds.

Δημιουργία, in vitro πολλαπλασιασμός και διαφοροποίηση ανθρώπινων επαγόμενων πολυδύναμων βλαστοκυττάρων

2016 ◽  
Author(s):  
Ιωάννα Βαρελά
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Rt Pcr ◽  
Induced Pluripotent

Η ανακάλυψη της μεθόδου του κυτταρικού επαναπρογραμματισμού ανθρώπινων δερματικών ινοβλαστών σε επαγόμενα πολυδύναμα βλαστοκύτταρα (induced pluripotent stem cells, iPSCs) το 2007 άνοιξε το δρόμο για τη μελέτη και την εξατομικευμένη θεραπεία πολλών χρόνιων νόσων. Επιδιώξαμε να δημιουργήσουμε iPS - κυτταρικές σειρές επαναπρογραμματίζοντας μεσεγχυματικά στρωματικά κύτταρα (mesenchymal stromal cells, MSCs) μυελού των οστών, μέσω μιας μεθόδου επαναπρογραμματισμού χωρίς ενσωμάτωση γονιδίων στο γενετικό υλικό των κυττάρων. Δερματικοί ινοβλάστες από φυσιολογικούς δότες και μεσεγχυματικά στρωματικά κύτταρα μυελού των οστών από φυσιολογικό δότη μεταμόσχευσης μυελού των οστών και από ασθενή με β-Μεσογειακή αναιμία (β-ΜΑ) διαμολύνθηκαν, μέσω λιποσωματικών φορέων, με συνθετικά mRNA που κωδικοποιούν τους μεταγραφικούς παράγοντες Oct4, Klf4, Sox2, Lin28, c-Myc. Στη συνέχεια, τα κύτταρα ελέγχθηκαν σε καλλιέργειες για τον σχηματισμό αποικιών πολυδύναμων βλαστοκυττάρων. Οι αποικίες απομονώθηκαν και με συνεχείς ανακαλλιέργειες δημιουργήθηκαν κυτταρικές σειρές, οι οποίες εξετάστηκαν για την πολυδυναμία τους με μεθόδους ανίχνευσης της έκφρασης των μεταγραφικών παραγόντων πολυδυναμίας (κυτταρομετρία ροής, RT-PCR, μελέτη του μεταγραφώματος με RNA μικροσυστοιχίες). Ως θετικός μάρτυρας και μέτρο σύγκρισης χρησιμοποιήθηκε πολύ καλά χαρακτηρισμένη εμβρυονική σειρά πολυδύναμων βλαστοκυττάρων. Οι iPS-κυτταρικές σειρές μελετήθηκαν, επίσης, ως προς τη λειτουργική τους πολυδυναμία με τον έλεγχο της ικανότητας τους να δημιουργούν in vitro εμβρυϊκά σωματίδια και in vivo τερατώματα μετά από υποδόρια εμφύτευση τους σε ανοσοανεπαρκείς ποντικούς, και ως προς τη δυνατότητα διαφοροποίησής τους σε αιμοποιητικά προγονικά κύτταρα. Η γενετική σταθερότητα των κυτταρικών σειρών ελέγχθηκε με DNA μικροσυστοιχίες συγκριτικού γονιδιωματικού υβριδισμού (aCGH). Απομονώθηκαν 3 iPS κυτταρικές σειρές από κάθε δείγμα κυττάρων, οι οποίες εμφανίζουν μεταγράφωμα πανομοιότυπο με εκείνο των πολυδύναμων εμβρυονικών βλαστοκυττάρων και. δημιουργούν εμβρυϊκά σωματίδια in vitro και τερατώματα in vivo, τα οποία αποτελούνται από ιστούς καταγωγής και από τα τρία βλαστικά δέρματα. Τα iPSCs των κυτταρικών σειρών πολλαπλασιάζονται για μεγάλο χρονικό διάστημα χωρίς μορφολογικές ενδείξες διαφοροποίησης. Με τη μέθοδο aCGH, στις iPS κυτταρικές σειρές μετά την 10η ανακαλλιέργεια ανιχνεύθηκαν πολυμορφισμοί στον αριθμό αντιγράφων (CNVs), τα οποία ήταν ελλείμματα μεγέθους περίπου 3 Mb. Η διαφοροποίηση των iPSCs σε αιμοποιητικά προγονικά κύτταρα οδήγησε στην παραγωγή CD34+ κυττάρων σε ποσοστό 8-10% των παραχθέντων κυττάρων με ασθενούς έντασης συνέκφραση του CD45, προσομοιάζοντας στο αιμαγγειακό στελεχιαίο κύτταρο. Στην παρούσα διατριβή παρουσιάζεται, για πρώτη φορά στην Ελλάδα, εξ όσων γνωρίζουμε, η τεχνολογία παραγωγής ανθρώπινων iPSCs με μια ασφαλή και αξιόπιστη μέθοδο. Οι iPSCs-κυτταρικές σειρές μπορεί να χρησιμοποιηθούν στη μελέτη ασθενειών, στον έλεγχο φαρμάκων και στην ανάπτυξη πρωτοκόλλων ιστικής μηχανικής και κυτταρικής θεραπείας.

Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells

2016 ◽  
Vol 61 (2) ◽  
pp. 154-167 ◽  
Author(s):  
Chunyu Bai ◽  
Xiangchen Li ◽  
Yuhua Gao ◽  
Ziao Yuan ◽  
Pengfei Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Reprogramming Efficiency ◽  
Induced Pluripotent

scholarly journals Potential of cardiac stem/progenitor cells and induced pluripotent stem cells for cardiac repair in ischaemic heart disease

2013 ◽  
Vol 125 (7) ◽  
pp. 319-327 ◽  
Author(s):  
Wei Eric Wang ◽  
Xiongwen Chen ◽  
Steven R. Houser ◽  
Chunyu Zeng
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Heart Disease ◽  
Cell Therapy ◽  
Induced Pluripotent Stem Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Autologous Transplantation ◽  
Cardiac Repair ◽  
Induced Pluripotent

Stem cell therapy has emerged as a promising strategy for cardiac and vascular repair. The ultimate goal is to rebuild functional myocardium by transplanting exogenous stem cells or by activating native stem cells to induce endogenous repair. CS/PCs (cardiac stem/progenitor cells) are one type of adult stem cell with the potential to differentiate into cardiac lineages (cardiomyocytes, smooth muscle cells and endothelial cells). iPSCs (induced pluripotent stem cells) also have the capacity to differentiate into necessary cells to rebuild injured cardiac tissue. Both types of stem cells have brought promise for cardiac repair. The present review summarizes recent advances in cardiac cell therapy based on these two cell sources and discusses the advantages and limitations of each candidate. We conclude that, although both types of stem cells can be considered for autologous transplantation with promising outcomes in animal models, CS/PCs have advanced more in their clinical application because iPSCs and their derivatives possess inherent obstacles for clinical use. Further studies are needed to move cell therapy forward for the treatment of heart disease.

scholarly journals Technological Progress in Generation of Induced Pluripotent Stem Cells for Clinical Applications

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Seung-Ick Oh ◽  
Chang Kyu Lee ◽  
Kyung Jin Cho ◽  
Kyung-Ok Lee ◽  
Ssang-Goo Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Chromosomal Abnormalities ◽  
Patient Specific ◽  
Current State ◽  
Reprogramming Efficiency ◽  
Induced Pluripotent ◽  
Viral Dna Integration ◽  
Medical Interest

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is achieved by viral-mediated transduction of defined transcription factors. Generation of iPSCs is of great medical interest as they have the potential to be a source of patient-specific cells. For the eventual goal of clinical application, it is necessary to overcome the limitations of low reprogramming efficiency and chromosomal abnormalities due to viral DNA integration. In this paper, we summarize the current state of reprogramming technology for generation of iPSCs and also discuss potential approaches to the development of safe iPSCs for personalized cell-based replacement therapy.

scholarly journals 188 MicroRNA characterization in equine induced pluripotent stem cells

2019 ◽  
Vol 31 (1) ◽  
pp. 218
Author(s):  
L. N. Moro ◽  
G. Amin ◽  
V. Furmento ◽  
A. Waisman ◽  
G. Neiman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Seed Region ◽  
Seed Sequence ◽  
Rt Pcr ◽  
Pluripotent Cells ◽  
Mesenchymal Transition ◽  
Mrna Targets ◽  
Induced Pluripotent

Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. A single microRNA can regulate the expression of hundreds of mRNA targets, a property given by a short sequence (called “seed”) in positions 2 to 8 from the 5′ end that is complementary to the 3′ untranslated region (UTR) tail of specific mRNAs. We aimed to generate horse induced pluripotent stem cells (iPSC), characterise them, and evaluate the expression of different microRNAs (miR-302a, b, c, d, miR-205, miR-145, miR-9, miR-96, miR-125b, and miR-296) in pluripotency and differentiation. Both cell states were evaluated (pluripotency and differentiation) in order to understand more deeply the complex network of transcriptional regulation in different contexts but with the same genomic background. Two equine iPSC lines (named L2 and L3) were characterised after the reprogramming of equine fibroblasts with the 4 human Yamanaka factors (OCT-4, SOX-2, c-MYC, KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG, and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2, and c-MYC. In vitro differentiation to embryo bodies (EB) showed the capacity of the iPSC to differentiate into ectodermal, endodermal, and mesodermal phenotypes. MicroRNA expression was analysed by quantitative RT-PCR and resulted in higher expression of the miR-302 family, miR-9, and miR-96 in L2 and L3v. fibroblasts (P ≤ 0.05), as previously shown in human pluripotent cells. Moreover, down-regulation of miR-145 and miR-205 was observed. After differentiation to EB, greater expression of miR-96 was observed in the EB compared with iPSC, and the expression of miR-205 was induced but only in the EB-L2. In addition, we performed in silico analysis of horse and human microRNAs. First, we compared the horse-miR-302/367 cluster with the human-miR-302/367 cluster and determined a 75% homology between them. Moreover, the seed region of the horse-miR-302 family resulted complementary to the 3′ UTR of horse cell cycle regulator genes CDK2, CYCLIN D1, and E2F1, and to the 3′ UTR of the RHOC gene, which is involved in the epithelial-mesenchymal transition. The miR-145 seed sequence was complementary to the 3′ UTR region of the OCT-4 and KLF-4 horse genes. With respect to miR-9 and miR-96, the seed sequence of these genes were complementary to the HES1 and PAX-6 genes. In all cases, the same gene targets were previously demonstrated in humans. In conclusion, we report the generation and characterization of equine iPSC and determined for the first time the expression of microRNAs in equine pluripotent cells. Moreover, several results led us to think that the horse microRNAs evaluated herein are highly conserved in sequence and function with respect to the human species. It will now be necessary to generate directed differentiations to derivatives of the 3 germ layers in order to strengthen our results. This is the first report to evaluate the expression and possible targets of microRNAs in pluripotent cells from domestic animals.

337 PROMOTER-DEPENDENT SILENCING OF REPROGRAMMING TRANSCRIPTION FACTORS IN MOUSE INDUCED PLURIPOTENT STEM CELLS PRODUCED WITH SLEEPING BEAUTY TRANSPOSON VECTORS

2015 ◽  
Vol 27 (1) ◽  
pp. 257
Author(s):  
S. G. Petkov ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Sleeping Beauty ◽  
Rt Pcr ◽  
Induced Pluripotent ◽  
Pluripotency Genes

Epigenetic silencing of the transgenes has been considered a prerequisite for complete reprogramming of mouse somatic cells to induced pluripotent stem cells (miPSC). Here, we examined the activity status of the reprogramming transcription factors in miPSC produced with Sleeping Beauty (SB) transposon vectors carrying expression cassettes with the porcine OCT4, SOX2, c-MYC, and KLF4 (pOSMK) under the control of doxycycline (DOX)-inducible (TetO) or constitutive (CAG) promoters. Mouse embryo fibroblasts (MEF) were electroporated with SB-TetO-rTA-SV40pA-TetO-pOSMK-IRES-tdTomato-bGHpA (TetO group) or with SB-loxP-CAG-pOSMK-IRES-tdTomato-SV40pA-loxP (CAG group) together with SB100x (SB transposase). The cells were cultured on mitotically inactivated MEF feeders with DMEM supplemented with 20% knockout serum replacement, 2 mM l-glutamine, penicillin-streptomycin, nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1000 U mL–1 of ESGRO, and 5 µg mL–1 of DOX. The miPSC colonies were individually picked, disaggregated to single cells, and propagated further under the same culture conditions. Three cell lines from each experimental group were examined for pluripotency characteristics, and the activity of the transgenes was monitored by the presence of tdTomato fluorescence and by RT-PCR. The miPSC produced with TetO vector silenced the transgene expression within 11 days post-transfection (in the presence of DOX) and upregulated the endogenous pluripotency genes Oct4, Sox2, Nanog, Rex1, and Utf1. These cells showed typical miPSC morphology and ability to differentiate into cells from the 3 primary germ layers in vitro and in vivo (teratomas). At the same time, the miPSC from the CAG group did not silence the transgenes even after 20 passages of continuous propagation, although they upregulated the endogenous pluripotency genes similarly to the TetO group. Moreover, these cells also showed ability to differentiate in vitro into cells from the 3 germ layers (contracting cardiac myocytes, neurons, epithelia) expressing differentiation markers Afp, Sox17, Gata4, Gata6, cardiac troponin, nestin, and PGP 9.5. Following Cre-mediated excision of the reprogramming cassette, the miPSC from the CAG group continued to self-renew and the expression of pluripotency markers Oct4, Sox2, Nanog, and Rex1 did not change significantly, as evidenced by real-time RT PCR (all P > 0.1), showing that these cells were not dependent on the transgenes for maintaining their pluripotency characteristics. Currently, we are investigating the ability of the miPSC from the CAG group to differentiate in vivo by producing teratomas and chimeras. The results from our preliminary investigations suggest that porcine transcription factors can be used for production of miPSC and that the silencing of the reprogramming transcription factors in miPSC is promoter-dependent, but may not be absolutely necessary for complete reprogramming to pluripotency.

scholarly journals Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells

Stem Cells ◽  
2016 ◽  
Vol 34 (3) ◽  
pp. 581-587 ◽  
Author(s):  
Álvaro Muñoz-López ◽  
Eddy. H.J. van Roon ◽  
Damià Romero-Moya ◽  
Belén López-Millan ◽  
Ronald W. Stam ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human B Cells ◽  
Reprogramming Efficiency ◽  
Induced Pluripotent
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