118 INDUCING PLURIPOTENCY IN SOMATIC CELLS FROM THE SNOW LEOPARD (PANTHERA UNCIA), AN ENDANGERED FELID

2012 ◽  
Vol 24 (1) ◽  
pp. 171
Author(s):  
R. Verma ◽  
M. Holland ◽  
P. Smith ◽  
P. Verma
Keyword(s):  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Retroviral Vectors ◽  
Ips Cells ◽  
Transduction Efficiency ◽  
Snow Leopard ◽  
Ips Cell ◽  
Induced Pluripotency ◽  
Panthera Uncia

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMX) encoding either 4 (Oct-4, Sox-2, Klf-4 and cMyc) or 5 (Oct-4, Sox-2, Klf-4, cMyc and Nanog) human transcription factors. Transduction efficiency of the retrovirus was ascertained using pMX-green fluorescent protein transgene expression and averaged 96% from 3 repeated experiments. The reprogramming efficiency of initial colony formation was 0.000308% (37/120 000 cells plated) for 4-factor induction compared with 0.000517% (62/120 000) for 5-factor induction. Transduction with 4 factors resulted in the formation of small colonies of cells, which could not be maintained for more than 4 passages (P4). However, addition of Nanog to the transfection cocktail produced stable iPS cell colonies, which formed as early as Day 3. Colonies of cells were selected at Day 5 and expanded in vitro on mouse embryonic fibroblast feeder cells. The resulting cell line was positive for alkaline phosphatase, Oct-4, Nanog and stage-specific embryonic antigen-4 at P14. Also, RT-PCR confirmed that endogenous Oct-4 and Nanog were expressed by snow leopard iPS cells from P4; although all 5 human transgenes were transcribed at P4, Oct-4, Sox-2 and Nanog transgenes were silenced as early as P14, suggesting that reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the 3 germ layers. This study describes the first derivation of iPS cells from the endangered snow leopard and is also the first report on induced pluripotency in felid species. Our results demonstrate that addition of Nanog to the reprogramming cocktail was essential for derivation of iPS lines in this felid and that iPS cells provide a unique source of pluripotent cells with utility in conservation for cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

2012 ◽  
Vol 77 (1) ◽  
pp. 220-228.e2 ◽  
Author(s):  
R. Verma ◽  
M.K. Holland ◽  
P. Temple-Smith ◽  
P.J. Verma
Keyword(s):  
Somatic Cells ◽  
Snow Leopard ◽  
Panthera Uncia

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.

Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2534-2534
Author(s):  
Akira Niwa ◽  
Tomoki Fukatsu ◽  
Katsutsugu Umeda ◽  
Itaru Kato ◽  
Hiromi Sakai ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Specific Antigen ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Oxygen Carriers ◽  
Developmental Pathway ◽  
Ips Cell

Abstract Abstract 2534 Poster Board II-511 Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production. Disclosures: No relevant conflicts of interest to declare.

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.

Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation

Blood ◽  
2010 ◽  
Vol 116 (3) ◽  
pp. 386-395 ◽  
Author(s):  
Hyun-Jai Cho ◽  
Choon-Soo Lee ◽  
Yoo-Wook Kwon ◽  
Jae Seung Paek ◽  
Sun-Hee Lee ◽  
...  
Keyword(s):  
Prolonged Exposure ◽  
Genetic Manipulation ◽  
Es Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Cell Protein ◽  
Generation Process ◽  
Es Cell

Abstract The concept of reprogramming of somatic cells has opened a new era in regenerative medicine. Transduction of defined factors has successfully achieved pluripotency. However, during the generation process of induced pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity, which limits further application. We report that that a single transfer of embryonic stem (ES) cell–derived proteins into primarily cultured adult mouse fibroblasts, rather than repeated transfer or prolonged exposure to materials, can achieve full reprogramming up to the pluripotent state without the forced expression of ectopic transgenes. During the process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. We verified that protein-based reprogramming was neither by the contamination of protein donor ES cell nor by DNA/RNA from donor ES cell. Protein-iPS cells were biologically and functionally very similar to ES cells and differentiated into 3 germ layers in vitro. Furthermore, protein-iPS cells possessed in vivo differentiation (well-differentiated teratoma formation) and development (chimeric mice generation and a tetraploid blastocyst complementation) potentials. Our results provide an alternative and safe strategy for the reprogramming of somatic cells that can be used to facilitate pluripotent stem cell–based cell therapy.

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.

Generation of Blood Cells from Human Ips Cells in Vitro through the Hematopoietic Progenitors Concentrated within the Unique Structures, Ips-Sac.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1992-1992 ◽  
Author(s):  
Naoya Takayama ◽  
Koji Eto ◽  
Hiromitsu Nakauchi ◽  
Shinya Yamanaka
Keyword(s):  
Cell Lines ◽  
Blood Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Dermal Fibroblasts ◽  
Hematopoietic Progenitors ◽  
Transfusion Therapy ◽  
Ips Cell ◽  
Human Ips Cells

Abstract Human embryonic stem cells (hESCs) are proposed as an alternative source for transfusion therapy or studies of hematopoiesis. We have recently established an in vitro culture system whereby hESCs can be differentiated into hematopoietic progenitors within the ‘unique sac-like structures’ (ES-sacs), that are able to produce megakaryocytes and platelets (Takayama et al., Blood, 111, 5298–306, 2008). However there is a little concern that repetitive transfusion with same human ESC-derived platelets may induce immunological rejection against transfused platelets expressing allogenic HLA. Meanwhile, induced pluripotent stem (iPS) cells established from donor with identical HLA are well known as a potential and given source on platelet transfusion devoid of rejection. To examine if human iPS cells could generate platelets as well as from hESCs, we utilized 3 different human iPS cell lines; two were induced by transduction of 4 genes (Oct3/4, Klf4, Sox2, and c-Myc) in adult dermal fibroblasts, and one was by 3 genes without c-Myc. Sac-like structures (iPS-sac), inducible from 3 iPS cell lines, concentrated hematopoietic progenitors that expressed early hemato-endothelial markers, such as CD34, CD31, CD41a (integrin αIIb) and CD45. These progenitors were able to form hematopoietic colonies in semi-solid culture and differentiate into several blood cells including leukocytes, erythrocytes or platelets. Of these, obtained platelets responded to agonist stimulation, in which the function was as much as human ESC-derived platelets, as evidenced by PAC-1 binding with activated αIIbβ3 integrin or full spreading onto fibrinogen. These results collectively indicated that human dermal fibroblasts could generate functional and mature hematopoietic cells through the reprogramming process and this method may be useful for basic studies of hematopoietic disorders and clinical therapy in the future.

Abstract 244: A Novel Method for the Generation of Induced Pluripotent Stem Cells From Human Peripheral Blood

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Han-Mo Yang ◽  
Ju-Young Kim ◽  
Yoo-Wook Kwon ◽  
Hyo-Soo Kim
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Peripheral Blood ◽  
High Efficiency ◽  
Human Peripheral Blood ◽  
Gene Transfection ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Ips Cell ◽  
Induced Pluripotent

Background: In terms of the generation of induced pluripotent stem(iPS) cells, one of the important issues for clinical applications is cell source. Human peripheral blood is one of the easily accessible cell sources. However, isolated peripheral blood cells have shown low gene transfection efficiency and inconveniences requiring specific methods to isolate. Here, we report a novel population of peripheral blood-derived stem cells, which can be easily reprogrammed to iPS cells. Methods and Results: We cultured peripheral blood mononuclear cells (PBMC) from human peripheral blood and seeded on the fibronectin-coated plate. We observed adherent cells from as early as 5 days after the start of culture and those cells gradually formed colonies. We were able to isolate these cells with very high efficiency. Furthermore, we have also confirmed that these cells can be differentiated to osteogenic, adipogenic, and myogenic-lineage cells. Therefore, we named these cells circulating multipotent adult stem cell. We were successful in generating iPS cells with these cells. These cells showed enhanced efficiency of gene transduction, compared to the human dermal fibroblast. We obtained reprogrammed colonies in 8 days after 4 factor virus transduction without feeder cells. We identified our iPS cells had similar features to embryonic stem cell in morphology, gene expression, epigenetic state and ability to differentiate into the three germ layers. We obtained more than 46 iPS cell lines from PBMC of patients with cardiovascular disease and normal volunteers. Conclusions: Our study showed new methods to isolate stem cells from peripheral blood and to generate iPS cells with high efficacy. This result suggests that our new approach could be one of ideal methods for clinical application of iPS cells in future.

Patient-Specific Pluripotent Stem Cells for Hematologic Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-40-SCI-40
Author(s):  
George Q. Daley
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Patient Specific ◽  
Direct Reprogramming ◽  
Disease Specific

Abstract Abstract SCI-40 Pluripotent stem cells can be isolated from embryos (embryonic stem cells; ES cells) or generated by direct reprogramming of somatic cells (induced pluripotent stem cells; iPS cells). Both types can be differentiated into a multitude of cell lineages to serve disease research and cell replacement therapies. Additionally, genetically matched pluripotent stem cells generated via nuclear transfer (ntES cells), parthenogenesis (pES cells), or direct reprogramming (iPS cells) are a possible source of histocompatible cells and tissues for transplantation. We have used customized ntES cells to repair genetic immunodeficiency in mice (Rideout et al., Cell 2002); however, generation of ES cells by nuclear transfer remains inefficient, and to date has not been achieved with human cells. We have also generated ES cells with defined histocompatibility loci by direct parthenogenetic activation of the unfertilized oocyte (Kim et al., Science 2007). Compared to ES cell lines from fertilized embryos, pES cells display comparable in vitro hematopoietic activity, but appear compromised in repopulating hematopoiesis in irradiated adult mouse recipients. We are currently comparing the performance of ntES, pES, and iPS cells in murine models of thalassemia. We have generated human iPS cells by direct reprogramming of human somatic cells with OCT4, SOX2, MYC, and KLF4 (Park et al., Nature 2008), and have generated disease-specific iPS cells from patients with a number of hematologic conditions (Park et al., Cell 2008; Agarwal et al., submitted). Applications of disease-specific cells for investigating the mechanisms of reprogramming and for probing aspects of human bone marrow disorders will be discussed. Disclosures Daley: iPierian: Consultancy, Equity Ownership; Epizyme: Consultancy; Solasia: Consultancy; MPM Capital: Consultancy.

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.

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