Analysis of Viral Integration Sites in Human Induced Pluripotent Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1485-1485
Author(s):  
Thomas Winkler ◽  
Amy R Cantelina ◽  
Jean-Yves Metais ◽  
Xiuli Xu ◽  
Anh-Dao Nguyen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Transfer ◽  
Cell Lines ◽  
Chromosomal Location ◽  
Somatic Cells ◽  
Ips Cells ◽  
Ips Cell ◽  
Integration Sites ◽  
Equity Ownership ◽  
Induced Pluripotent

Abstract Abstract 1485 Poster Board I-508 The recently discovered approach for the direct reprogramming of somatic cells into induced pluripotent stem (IPS) cells by expression of defined transcription factors may provide new approaches for regenerative medicine, gene therapy and drug screening. Successful reprogramming currently requires at least temporary expression of one to four different transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog and Lin28) in the targeted cells. Non-viral based reprogramming technologies have been reported, but expression of the reprogramming factors after γ-retroviral or lentiviral gene transfer remains the most efficient and commonly used approach. Since the reprogramming frequency is consistently low in these studies, it has been speculated that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. Here we present for the first time an extensive analysis of the lentiviral integration profile in human IPS-cells. We analysed the IS of 8 IPS cell lines derived from either human fetal fibroblasts (IMR90) or newborn foreskin fibroblasts (FS) after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28, using linear amplification-mediated PCR (LAM-PCR). With 5 to15 IS per individual IPS clone we identified a total of 78 independent IS. Finally we assigned 75 IS to a unique chromosomal location. In addition to LAM-PCR, we confirmed the total number of IS via Southern blot. Interestingly, in 6 of 8 IPS clones some of these IS were found in pairs, integrated into the same chromosomal location within 4 base pairs of each other. This integration pattern has not been detected in our previous analysis of 702 IS in rhesus macaques transplanted with CD34+ cells transduced with retroviral vectors. Of the 75 valid IS 53 (70.7%) could be mapped to a gene-coding region, 52 located in introns and 1 in an exon, annotated in a human reference sequence in the UCSC Genome Browser RefSeq Genes track. The different IPS-clones had no integration site in common. To investigate the impact of integration on the regulation of vector targeted genes we analyzed the mRNA expression profiles using available microarray data from these clones. Out of 46 evaluable genes only two (WDR66 and MYST2 in clone IMR90-2, p<0.0001) were significantly over-expressed. The expression of two genes in clone FS-1 (ACVR2A p=0.01, RAF1 p=0.02) and one in FS-2 (KIAA0528, p=0.03) was decreased compared to the expression data of all other clones combined. In summary our data suggest that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes. Furthermore, identification of the insertion profile of the IPS cell clones IMR90-1 and -4 as well as FS-1 will be useful to other researchers using these cell lines distributed by the Wisconsin International Stem Cell (WISC) bank. Disclosures: Antosiewicz-Bourget: Cellular Dynamics International: Consultancy, Equity Ownership. Thomson: Cellular Dynamics International: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dunbar: ASH: Honoraria.

Targeted Gene Correction of Glanzmann Thrombasthenia Induced Pluripotent Stem Cells Restores Surface Expression and Fibrinogen Binding of Integrin αIIbβ3,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4173-4173
Author(s):  
Spencer Sullivan ◽  
Jason A. Mills ◽  
Li Zhai ◽  
Prasuna Paluru ◽  
Guohua Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Cell Lines ◽  
Surface Expression ◽  
Ips Cells ◽  
Ips Cell ◽  
Glanzmann Thrombasthenia ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Induced Pluripotent

Abstract Abstract 4173 Glanzmann Thrombasthenia (GT) is a rare, autosomal recessive disorder resulting from an absence of functional platelet integrin αIIbβ3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. It is a model of an inherited platelet disorder that might benefit from corrective gene therapy. Treatment options for GT are limited and largely supportive. They include anti-fibrinolytics, activated factor VII, platelet transfusions, and bone marrow transplantation. Recent gene therapy research in a canine model for GT demonstrated that lentiviral transduction of mobilized hematopoietic stem cells could restore 6% αIIbβ3 receptors in thrombasthenic canine platelets relative to wild type (WT) canine platelets. As an alternative gene therapy strategy, we generated induced pluripotent stem (iPS) cell lines from the peripheral blood of two patients with GT and examined whether a megakaryocyte-specific promoter driving αIIb cDNA expression within the AAVS1 safe harbor locus could ameliorate the GT phenotype in iPS cell-derived megakaryocytes. Patient 1 is a compound heterozygote for αIIb with the following two missense mutations: exon 2 c.331T>C (p.L100P) and exon 5 c.607G>A (p.S192N). Patient 2 is homozygous for a c.818G>A (p.G273D) mutation adjacent to the first calcium-binding domain of αIIb, leading to impaired intracellular transport of αIIbβ3. Both patients express <5% αIIbβ3 on the surface of their platelets. Peripheral blood mononuclear cells from both GT patients and WT controls were efficiently reprogrammed to pluripotency using a doxycycline-inducible polycistronic lentivirus containing OCT4, KLF4, SOX2, and CMYC. Transgene constructs using a murine GPIbα promoter driving either a green fluorescent protein (GFP) reporter or αIIb cDNA were inserted into a gene-targeting vector specific for the first intron of AAVS1, a locus amenable to gene targeting and resistant to transgene silencing in human iPS cells. The GPIbα-driven GFP transgene was efficiently targeted into AAVS1 in WT iPS cells using zinc finger nuclease-mediated homologous recombination, as was the αIIb construct into GT iPS cell lines. PCR and Southern blot analyses confirmed single, non-random, transgene integrations. The iPS cells were differentiated into megakaryocytes using a feeder-free/serum-free adherent monolayer protocol and analyzed by flow cytometry. GFP, along with endogenous CD41 (αIIb), was initially expressed in primitive WT hematopoietic progenitor cells. GFP expression was lost in erythrocytes and myeloid cells, but maintained in CD41+/CD42+ megakaryocytes, demonstrating that this transgenic construct mirrors endogenous CD41 expression. The GT phenotype was confirmed in megakaryocytes derived from patient iPS cells, showing loss of αIIbβ3 expression. When compared to WT iPS cell-derived megakaryocytes, gene-corrected GT iPS cell-derived megakaryocytes showed >50% and >70% αIIbβ3 surface expression for patients 1 and 2, respectively. Both patients' iPS cell-derived megakaryocytes also demonstrated fibrinogen binding upon thrombin activation. This is the first report of the generation and genetic correction of iPS cell lines from patients with a disease affecting platelet function. These findings suggest that this GPIbα-promoter construct targeted to the AAVS1 locus drives megakaryocyte-specific expression at a therapeutically significant level, which offers the possibility of correcting severe inherited platelet disorders beginning with iPS cells derived from these affected individuals. Disclosures: Lambert: Cangene: Honoraria.

Reprogramming of Leukemic and Pre-Leukemic Cells from Primary Human De Novo Acute Myeloid Leukemia Samples into Induced Pluripotent Stem (iPS) Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1862-1862
Author(s):  
David Spencer ◽  
Daniel R. George ◽  
Jeffery M. Klco ◽  
Timothy J. Ley
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Ips Cells ◽  
Ips Cell ◽  
A Cell ◽  
Primary Bone ◽  
Induced Pluripotent ◽  
Primary Bone Marrow

Abstract Somatic reprogramming captures the mutations present in individual cells and can yield induced pluripotent stem (iPS) cells that can be used to study these mutations in their native genetic context. iPS cells have been made using a variety of primary tissues and established cell lines, but to date there have been few examples of somatic reprogramming using primary cancer samples. Some studies have reported iPS cell generation using samples from patients with myeloproliferative neoplasms (Ye Z Blood 2009, Hosoi M Exp. Hematol. 2014), and another study successfully reprogrammed primary bone marrow cells from patients with myelodysplastic syndromes (MDS) (Kotini AG Nat. Biotech. 2015). However, it is not yet clear whether fully transformed human myeloid leukemia cells can be reprogrammed to an undifferentiated state. Here we describe the results of reprogramming experiments and subsequent genetic characterization of iPS clones produced from primary bone marrow and peripheral blood samples from adult human de novo AML patients. Our reprogramming approach involved in vitro culture of primary cells on Hs27 stroma with hematopoietic cytokines for 3-7 days, followed by transfer of 250,000 cells to stroma-free conditions for transduction with nonintegrating Sendai viruses expressing cMyc, OCT3/4, KLF4, and SOX2. Cells were then returned to AML culture conditions with stroma for 2-4 days before plating on mouse embryonic fibroblasts (MEF) in human embryonic stem (ES) cell media for 2-6 weeks. Individual clusters of cells with undifferentiated iPS cell colony morphology were then picked and expanded on either MEFs or feeder-free conditions. We performed 21 transductions using 8 peripheral blood and 13 bone marrow samples from 16 AML patients (i.e., multiple samples were attempted for some AMLs), which yielded 65 iPS clones from 9 of the 16 AML patients (56%) that were successfully expanded for genomic analysis. The remaining AMLs either produced no colonies (N=5), or clones that failed to expand after transferring from the original plate (N=2). Initial analysis of representative iPS clones (N=4) via flow cytometry demonstrated expression of the pluripotency markers SSEA-4 and TRA-1-60. Additional experiments to assess the pluripotency of these iPS lines are currently underway, including analysis of all clones via flow cytometry, RNA-sequencing, and teratoma formation assays. To determine the relationship between each iPS clone and the original AML samples used for reprogramming, we performed targeted sequencing for all somatic mutations identified from either whole-genome or exome sequencing. Analysis of each iPS clone for multiple patient-specific AML mutations (range 12-683) demonstrated that the reprogrammed cells were derived from 1 of 3 distinct cell types, depending on the sample. The most common type (N=1, 1, 3, 10, and 12 clones from 5 AMLs) possessed virtually no AML mutations (Figure 1A), suggesting that reprogramming occurred in a cell population that was unrelated to the tumor. Another 24 clones from 2 AML samples (N=1 and N=23) contained a subset of the AML-associated mutations (Figure 1B), but lacked common AML mutations that are generally cooperating 'hits', such as NPM1, and FLT3; for these samples, reprogramming probably occurred in a cell that was ancestral to the AML founding clone (i.e., a pre-leukemic cell). The final group of 14 clones from 2 AMLs (N=7 for both samples) contained the majority of AML-associated mutations in those samples, including canonical mutations in IDH1 and IDH2, and mutations in DNMT3A and RUNX1 (Figure 1C), implying that reprogramming occurred in the most prevalent AML subclone in the sample. Remarkably, for AML samples that yielded >1 iPS clone (N=6), all the iPS clones had the same set of mutations, suggesting that some of the cells in the sample were more "fit" for reprogramming than others. In conclusion, we have generated iPS cell lines from 9 primary AML samples, several of which contain canonical AML mutations. In this study, the majority of the reprogramming events took place in rare cells from clones that were not the most abundant cells in the sample. However, in one case, all iPS clones were derived from the most prevalent AML subclone in the sample. Future study of these iPS cell lines will provide insights into epigenetic dysregulation in cancer, and of the functional consequences of the mutational combinations that were "captured" via reprogramming. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Blood ◽  
2009 ◽  
Vol 114 (27) ◽  
pp. 5473-5480 ◽  
Author(s):  
Zhaohui Ye ◽  
Huichun Zhan ◽  
Prashant Mali ◽  
Sarah Dowey ◽  
Donna M. Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Blood Cells ◽  
Ips Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Ips Cell ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Induced Pluripotent

Abstract Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

MODELING GENETIC Diseases through Reprogramming of HUMAN Amniotic Liquid Derived CELLS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4789-4789
Author(s):  
Noufissa Oudrhiri ◽  
Frank Yates ◽  
Olivier Feraud ◽  
Emilie Gobbo ◽  
Cecile BAS ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Sickle Cell Anemia ◽  
Genetic Diseases ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Ips Cell

Abstract Abstract 4789 Pluripotency and self-renewal, two key characteristics of induced pluripotent stem cells (IPS), make these cells ideally suited for modeling diseases in vitro and generating biological resources usable for drug screening and cell therapy. However, the reprogramming efficiency of somatic cells greatly varies according to the cell type, to the in vitro proliferation index, the number of passages and the age of the donor. Human amniotic liquid-derived cells (hALDC), collected during amniocentesis for the prenatal diagnosis of genetic diseases, represent an abundant source of primary cells. In preliminary experiments we have shown that hALDC expressed endogenous Oct4 and Sox2 proteins suggesting that could be readily amenable to reprogramming. To this end, we have used two strategies using either hALDC or neonatal fibroblasts: (1) lentivirus mediated gene transfer of OCT4, SOX2, LIN28, NANOG, (2) retroviruses mediated gene transfer of OCT4, SOX2, CMYC, KLF4 and (3) lentiviral transfer of OCT4, SOX2. hALDC transduced by these viruses were placed on MEF and b-FGF (10 ng/ml) with daily medium changes. One to three weeks after infection, typical human ES-like colonies could be picked up for expansion before being characterized. HALDC show an increased reprogramming potential with the [OCT4, SOX2, LIN28, NANOG] and [OCT4, SOX2] cocktails, when compared to reprogramming of neonatal fibroblasts. Twelve hALDC-derived-IPS cells were obtained from 12 different samples of amniotic fluid. All hALDC-IPS cell lines maintained a normal karyotype in culture and displayed the morphology and characteristics of human embryonic stem cells, including the surface expression of Tra-160, SSEA-3, SSEA-4, HESCA-1 and alkaline phosphatase, and formed multi-lineaged teratomas upon injection to NOD-SCID mice. Gene expression profiles of the IPS cell lines reveal a high correlation coefficient between hALDC-iPS cells and human embryonic stem cells, and a low correlation between hALDC-iPS and hALDC. When compared to hES cells H1, H9 and Cl01, these cell lines generated hematopoiesis with a variable efficiency in vitro. Amongst the hALDC-IPS cell lines generated by our laboratory (http://www.hescreg.eu/) four lines carry an inherited trisomy of chromosome 21, and three lines carry the homozygous “S” mutation in the beta-globin gene of sickle-cell anemia. All hALDC-IPS cell are currently banked at the Human Pluripotent Stem Cell Core Facility, France. In conclusion, hALDC can be rapidly and efficiently reprogrammed to pluripotency with a limited number of transgenes. Moreover, hALDC-IPS cell lines derived from patients can be used to modelize in vitro the phenotypic features of monogenic diseases such as sickle cell anemia or more complex, multifactorial disorders such as Down's syndrom. The ability to generate hematopoietic differentiation from these cell lines will facilitate the modelling of these hematopoietic disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular Mechanisms of Induced Pluripotency

Acta Naturae ◽  
2012 ◽  
Vol 4 (1) ◽  
pp. 12-22 ◽  
Author(s):  
I. A. Muchkaeva ◽  
E. B. Dashinimaev ◽  
V. V. Terskikh ◽  
Yu. V. Sukhanov ◽  
A. V. Vasiliev
Keyword(s):  
Transcription Factors ◽  
Small Molecules ◽  
Molecular Mechanisms ◽  
Somatic Cells ◽  
Ips Cells ◽  
Direct Reprogramming ◽  
Induced Pluripotency ◽  
Somatic Cell Reprogramming ◽  
Induced Pluripotent ◽  
Genome Modifications

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered.

scholarly journals Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors

Cell Research ◽  
2008 ◽  
Vol 18 (5) ◽  
pp. 600-603 ◽  
Author(s):  
Jing Liao ◽  
Zhao Wu ◽  
Ying Wang ◽  
Lu Cheng ◽  
Chun Cui ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Somatic Cells ◽  
Ips Cells ◽  
Induced Pluripotent

Reduced Production of Mature Neutrophils From Induced Pluripotent Stem Cells Derived From a Severe Congenital Neutropenia Patient with HAX1 Gene Deficiency

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2402-2402 ◽  
Author(s):  
Tatsuya Morishima ◽  
Ken-ichiro Watanabe ◽  
Akira Niwa ◽  
Takayuki Tanaka ◽  
Katsutsugu Umeda ◽  
...  
Keyword(s):  
Cell Lines ◽  
Normal Control ◽  
Annexin V ◽  
Ips Cells ◽  
Patient Specific ◽  
Es Cell ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Ips Cell ◽  
Induced Pluripotent

Abstract Abstract 2402 Induced pluripotent stem (iPS) cells are reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics. As iPS cells can be generated from somatic cells of patients with a certain disease, they are expected to be a novel model to study pathogenesis of various diseases. Recently, we established a neutrophil differentiation system from human iPS cells (Morishima T, et al. J Cell Physiol. 2011). In an attempt to apply the system to investigate pathophysiology of neutrophil-affected disorders, we generated iPS cells from a severe congenital neutropenia (SCN) patient with HAX1 gene deficiency. The patient was an 11-year-old boy with severe congenital neutropenia as well as developmental delay and epilepsy. DNA sequence analysis revealed HAX1 gene mutation in exon 2 (Matsubara K, et al. Haematologica. 2007). Four iPS cell lines were generated from skin fibroblasts of the patient by retroviral overexpression of the three or four transcription factors Oct3/4, Sox2, and Klf4, with or without c-Myc. These patient-derived iPS cell lines showed human ES cell like morphology and could be maintained under human ES cell culture condition. They also expressed typical human ES cell markers and were capable of differentiating into the cell lineages and tissues representing three germ layers by teratoma formation in vivo. These cells had normal karyotype and short tandem repeat analysis indicated that they were derived from parental skin fibroblast. DNA sequencing analysis of the iPS cell lines identified the same mutation carried in the parental skin fibroblasts, thus confirmed that we had established the HAX1 deficiency patient-specific iPS cells (HAX1-iPSCs). Next these HAX1-iPSCs and the healthy-person derived iPS cells were differentiated into neutrophils in vitro using feeder-free culture protocols established in our laboratory. In this culture system, small human iPS cell clumps were cultured on the matrigel-coated dish with recombinant cytokines and without any feeder cells and fetal calf serum. Around day 25 of culture, mature neutrophils were obtained as floating cells. Morphologically, the majority of HAX1-iPSCs-derived cells were classified into myeloblast or promyelocyte stage and there were only a few mature neutrophils. The proportion of mature neutrophils was only less than 10% in HAX1-iPSCs-derived cells whereas more than 40% in normal control. Flow cytometric analysis revealed that the percentage of immature CD34 positive cells was significantly higher and that of myeloid-committed CD11b positive cells was lower in the HAX1-iPSCs-derived cells than normal control. Immunocytochemical analysis for neutrophil specific granules showed that lactoferrin- and gelatinase-positive cells decreased in the HAX1-iPSCs-derived cells compared with normal control, confirming that HAX1-iPSCs-derived cells contained less mature neutrophils than normal control. Apoptosis assay by Annexin V staining revealed that HAX1-iPSCs-derived cells showed higher percentage of Annexin V-positive cells compared with normal control. Overall, these HAX1 deficiency patient-specific iPS cell lines recapitulate the hematological phenotype in the patient. These results indicate that patient-derived iPS cells provide us a novel disease model and make a contribution to the understanding of the pathophysiology of the diseases that affect neutrophils. Disclosures: No relevant conflicts of interest to declare.

292 GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS FROM VARIOUS GENETIC BACKGROUND BY SLEEPING BEAUTY TRANSPOSON-MEDIATED GENE TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 243 ◽  
Author(s):  
S. Muenthaisong ◽  
O. Ujhelly ◽  
E. Varga ◽  
A. C. Carstea ◽  
Z. Ivics ◽  
...  
Keyword(s):  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
Sleeping Beauty ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Differentiation Potential ◽  
Ips Cell ◽  
Sleeping Beauty Transposon ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cell technology allows the reprogramming of somatic cells to a pluripotent state; however, it requires viral gene transduction and permanent existence of the exogenous genes in the genome, which is a potential risk for abnormalities in the derived iPS cells. Recently, there was report that iPS cells have been made with piggyBack transposon. Here, we first reported that nonviral transfection of a Sleeping Beauty transposon, which comprises c-Myc, Klf-4, Oct3/4 (Pou5f1), and Sox-2, can reprogram mouse fibroblasts from 3 different genetic backgrounds: ICR (outbred), C57BL/6 (inbred), and F1 hybrid (C57BL/6 × DBA/2J), with parallel robust expression of all exogenous (c-Myc, Klf-4, Oct3/4, and Sox-2) and endogenous (e.g. Nanog) pluripotency genes. The iPS cells were cultured under standard conditions with promotion of differentiate by withdrawal of leukemia inhibitory factor. We chose 6 cloned of each line that exhibited characteristics typical for undifferentiated embryonic stem (ES) cell: ES-cell-like morphology, alkaline phosphatase positivity, and gene expression pattern [quantitative real-time PCR and immunofluorescence of ES cell markers (e.g. Oct-4, SSEA1, Nanog]. Furthermore, cells were able to form embryoid bodies and beat rhythmically and expressed cardiac markers assayed by immunofluorescence (e.g. cardiac Troponin T, desmin). In vivo testing of iPS cell lines for their developmental potential (diploid and tetraploid embryo complementation assay) is currently underway. The iPS cell lines generated from the ICR strain appeared the earliest in time (ICR-d11, F1 day-2 and Bl6-d12), with higher efficiency than colonies from the other 2 backgrounds. The differentiation potential of the iPS lines derived from the 3 genetic backgrounds was similar. Interestingly, the ICR-iPS lines had higher differentiation potential than did the ICR-ES cell lines: the rate of embryoid bodies forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. Our results suggest that the iPS technology provide a new tool to generate pluripotent stem cells from genetic backgrounds where good-quality ES cell generation is difficult. These studies provide new insights into virus-free iPS technology and contribute to defining future cell-based therapies, drug screening methods, and production of transgenic animals with genetically modified iPS cells. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205, and NKTH/KPI (Jedlik NKFP_07_1-ES2HEART-HU OM-00202-2007).

Human Embryonic Stem Cell-Specific Micrornas Promote Full Programming of Induced Pluripotent Stem Cells Derived From CD34+ Cord Blood Cells Stored Frozen for More Than 20 Years.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2622-2622
Author(s):  
Man Ryul Lee ◽  
Nutan Prasain ◽  
Young-June Kim ◽  
Mervin C. Yoder ◽  
Hal E. Broxmeyer
Keyword(s):  
Transcription Factors ◽  
Cord Blood ◽  
Production Efficiency ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Self Renewal ◽  
Ips Cell ◽  
Cd34 Cells ◽  
Induced Pluripotent ◽  
Hes Cells

Abstract Abstract 2622 Numerous somatic cells can be reprogrammed by ectopic expression of defined transcription factors including OCT4, SOX2, MYC, KLF4 (Yamanaka's factors), NANOG, and LIN28 to pluripotent cells, referred to as induced pluripotent stem (iPS) cells which can then be differentiated into a variety of somatic cell types. Production efficiency of iPS cells by these transcription factors is extremely low, and therefore a large portion of the cells remain unprogrammed or incompletely reprogrammed. Thus, identification of additional factors required for enhancing iPS cells production efficiency has been an intensive research subject. We generated iPS cell from CD34+ cells of human umbilical cord blood that had been frozen in an unseparated state for twenty plus years using lentiviruses expressing Yamanaka's factors. During the iPS cell production processes, we monitored cell surface expression of TRA1-60, a marker for human embryonic stem (hES) cells, within colonies using a live cell staining method. Three to four weeks after gene transduction, TRA1-60 positive cells emerged in about 5% of the colonies. So, we mechanically separated TRA1-60 negative cells from TRA1-60 positive cells in colonies. Further culture did not allow TRA1-60 negative cells to convert to TRA1-60 positive cells even after 10 passages, indicating that TRA1-60 negative cells were stable at an incompletely reprogrammed state. TRA1-60 negative cells were similar to hES cells in morphology and still demonstrated expression of exogenous Yamanaka's factors. TRA1-60 negative cells were distinct from TRA1-60 positive cells with regards to methylation pattern of OCT4 promoter regions and differentiation potential. In contrast to TRA1-60 positive cells, incompletely reprogrammed TRA1-60 negative cells lacked hESC-specific miRNAs (miR-302 and 371 clusters), which are known to be involved in controlling self-renewal and pluripotency of hES cells. We hypothesized that these miRNAs can promote a transition from incompletely to fully reprogrammed iPS cells. To test this hypothesis, we introduced the miRNA clusters of miR-302 and 371 using lentiviruses to TRA1-60 negative incompletely reprogrammed cell to determine whether these miRNAs could convert TRA-60 negative cells to TRA1-60 positive completely reprogrammed cells. Our results showed that these miRNAs were able to convert more than 10% of TRA1-60 negative incompletely reprogrammed cells to TRA1-60 positive iPS cells with characteristics of completely reprogrammed iPS cells, such as differentiation of three germ layers and acquisition of typical hES cell-specific cell cycling patterns with an unrestricted G1 to S phase transition. These results indicate that hES-specific miRNAs have a strong potential to promote partially reprogrammed cord blood CD34+ cells to iPS cells with extensive self-renewal capacity. Our study suggests that current techniques with low iPS cell production efficiency can be improved by ectopic overexpression of hES-specific miRNAs (miR-302 and371 clusters). Disclosures: Broxmeyer: Corduse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Development of a High-Efficacy Reprogramming Method for Generating Human Induced Pluripotent Stem (iPS) Cells from Pathologic and Senescent Somatic Cells

2020 ◽  
Vol 21 (18) ◽  
pp. 6764
Author(s):  
Naomichi Tanaka ◽  
Hidemasa Kato ◽  
Hiromi Tsuda ◽  
Yasunori Sato ◽  
Toshihiro Muramatsu ◽  
...  
Keyword(s):  
Heart Transplant ◽  
Somatic Cells ◽  
Heart Transplant Recipient ◽  
Ips Cells ◽  
Cell Reprogramming ◽  
Ips Cell ◽  
Cell Induction ◽  
Yamanaka Factors ◽  
Induced Pluripotent ◽  
Ips Cell Reprogramming

Induced pluripotent stem (iPS) cells are a type of artificial pluripotent stem cell induced by the epigenetic silencing of somatic cells by the Yamanaka factors. Advances in iPS cell reprogramming technology will allow aging or damaged cells to be replaced by a patient’s own rejuvenated cells. However, tissue that is senescent or pathologic has a relatively low reprogramming efficiency as compared with juvenile or robust tissue, resulting in incomplete reprogramming; iPS cells generated from such tissue types do not have sufficient differentiation ability and are therefore difficult to apply clinically. Here, we develop a new reprogramming method and examine it using myofibroblasts, which are pathologic somatic cells, from patient skin tissue and from each of the four heart chambers of a recipient heart in heart transplant surgery. By adjusting the type and amount of vectors containing transcriptional factors for iPS cell reprogramming, as well as adjusting the transfection load and culture medium, the efficiency of iPS cell induction from aged patient skin-derived fibroblasts was increased, and we successfully induced iPS cells from myocardial fibroblasts isolated from the pathologic heart of a heart transplant recipient.

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