325 CHARACTERIZATION OF PRIMED PORCINE PLURIPOTENT STEM CELL LINES DERIVED FROM VARIOUS ORIGINS INCLUDING iPS-NT

2015 ◽  
Vol 27 (1) ◽  
pp. 251
Author(s):  
E. Kim ◽  
C.-K. Lee ◽  
S.-H. Hyun
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Genetic Manipulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Donor Cell ◽  
Preclinical Research

Pigs are significant as a disease model in translational research. However, authentic porcine embryonic stem cells (ESC) have not yet been established showing limited capacities until now. In this study, a total of 7 primed ESC lines were derived from porcine embryos of various origins, including in vitro-fertilized (IVF), parthenogenetic activation (PA), and nuclear transfer (iPS-NT) from a donor cell with induced pluripotent stem cells (iPSC). We observed typical morphology, intensive alkaline phosphatase activity, and normal karyotype in all pESC lines. Also, the expression of pluripotency markers such as OCT4, Sox2, NANOG, SSEA4, TRA 1–60, and TRA 1–81 was shown in our pESC. We investigated expression of key markers of lineage commitment to confirm the differentiation potentials of the 7 cell lines to formation of EB and all 3 germ layers, such as AFP (endoderm), DESMIN (mesoderm), and CRABP2 (ectoderm) by RT-PCR and Cytokeratin 17 (endoderm), Desmin (mesoderm), and Vimentin (ectoderm) by immunofluorescence analysis. We also examined the XIST gene expression and nuclear H3K27me3 foci from all female cell lines for analysing epigenetic characteristics. Furthermore, we classified 2 colony types (normal and transformed colony) and 3 subpopulations of ES cells composed of transformed colonies with intrinsic morphological characteristics: petaloid rapidly self-renewing cells, small spindle-shaped cells, and large flattened cells. This result will help to approach the goal for establishing authentic naive pluripotent stem cells in pigs and it will make possible sophisticated genetic manipulation to create ideal animal models for preclinical research and studies of human diseases.This work was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Application of biomaterials to in vitro pluripotent stem cell disease modeling of the skeletal system

2016 ◽  
Vol 4 (20) ◽  
pp. 3482-3489 ◽  
Author(s):  
Giuliana E. Salazar-Noratto ◽  
Frank P. Barry ◽  
Robert E. Guldberg
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Embryonic Stem ◽  
Cell Disease ◽  
Skeletal System ◽  
System P ◽  
Induced Pluripotent

Disease-specific pluripotent stem cells can be derived through genetic manipulation of embryonic stem cells or by reprogramming somatic cells (induced pluripotent stem cells).

230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
S. Wang ◽  
X. Tang ◽  
Y. Niu ◽  
H. Chen ◽  
T. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Speed ◽  
Es Cells ◽  
Research Work ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

scholarly journals Generation of multipotent cell lines from a distinct population of male germ line stem cells

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 771-784 ◽  
Author(s):  
Fariborz Izadyar ◽  
Francis Pau ◽  
Joel Marh ◽  
Natalia Slepko ◽  
Tracy Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Complex Mixture ◽  
Neural Cells ◽  
Differentiation Potential

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Hepatic differentiation of pluripotent stem cells

2009 ◽  
Vol 390 (10) ◽  
Author(s):  
Komal Loya ◽  
Reto Eggenschwiler ◽  
Kinarm Ko ◽  
Malte Sgodda ◽  
Francoise André ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hepatic Differentiation ◽  
Alternative Source ◽  
Organ Specific

Abstract In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

scholarly journals Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

2015 ◽  
Vol 112 (18) ◽  
pp. E2337-E2346 ◽  
Author(s):  
Ying Yang ◽  
Katsuyuki Adachi ◽  
Megan A. Sheridan ◽  
Andrei P. Alexenko ◽  
Danny J. Schust ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Es Cells ◽  
Early Embryo ◽  
Extravillous Trophoblast ◽  
Cell Phenotype ◽  
Small Areas

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24–36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

Large Scale Generation of Functional Megakaryocytes From Human Embryonic Stem Cells (hESCs) Under Stromal-Free Conditions.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2540-2540
Author(s):  
Feng Li ◽  
Shi-Jiang Lu ◽  
Qiang Feng ◽  
Robert Lanza
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Es Cells ◽  
Scale Up ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Immunofluorescent Staining ◽  
Promising Alternative ◽  
Human Es Cells

Abstract Abstract 2540 Poster Board II-517 Platelets collected from donors have very limited shelf life and are increasingly needed for transfusions. In contrast to donor dependent cord blood or bone marrow CD34+ stem cells, hESCs are a promising alternative source for continuous in vitro production of platelets under controlled conditions. Current procedures for in vitro generation of megakaryocytes/platelets from hESCs are not efficient and require undefined animal stromal cells. We have developed a novel system to generate megakaryocytes (MKs) from human ES cells under serum and stromal-free conditions. In the current system, hESCs are directed towards megakaryocytes through distinct steps including embryoid body formation and hemangioblast development (Lu et al, Nature Methods, 4:501–509, 2007). A transient bi-potential cell population expressing both CD41a and CD235a markers has been identified at the end of hemangioblast culture. These cells are capable of generating both MKs and erythroid cells as demonstrated by FACS sorting and CFU assays. TPO, SCF and IL-11 are used to further direct MK differentiation of hemangioblasts derived from human ES cells in suspension culture. Currently, up to 2.5×107 MKs (CD41a+) can be generated from 1×106 hESCs, which is approximately 10 times more efficient than recently reported methods (Takayama et al Blood, 111(11):5298–5306, 2008). Without further purification, >90% of live cells from the suspension cultures are CD41a+ and the majority of these cells are also CD42b+ (>70%). These in vitro derived MK cells have morphological characteristics of mature, polyploid MKs as shown by Giemsa staining and immunofluorescent staining of vWF in cytoplasmic granules. Importantly, proplatelet forming cells are constantly observed at the late stage of MK culture indicating that MKs generated in this system are able to undergo terminal differentiation under feeder-free conditions. Platelet-like particles are also detected in culture media by FACS. When plated on OP9 cells, these MKs generate functional platelets that are responsive to thrombin stimulation. In summary, we have established a novel system for the generation of platelet-producing MKs from human ES cells that is suitable for scale up and future preclinical and clinical studies. Disclosures: Li: Stem Cell & Regenerative Medicine International: Employment. Lu:Stem Cell & Regenerative Medicine International: Employment. Feng:Stem Cell & Regenerative Medicine International: Employment. Lanza:Stem Cell & Regenerative Medicine International/Advanced Cell Technology, Inc: Employment.

scholarly journals Research Progress and Application Prospect of IPS Cells

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Ceng Yiwu
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Ips Cells ◽  
New Drugs ◽  
Theoretical Research ◽  
Research Progress ◽  
Ips Cell

Differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPS cells) by introducingspecific transcription factors. This technique avoids immune rejection and ethical problems in stem cell research.A great revolution in the fi eld of science. As with embryonic stem cells (ES cells), iPS cells are able to self-renewand maintain undiff erentiated state. In vitro, iPS cells can be induced to diff erentiate into a variety of mature cells,therefore, iPS cells in theoretical research and clinical applications are extremely valuable. IPS cell diff erentiationand transplantation in the treatment of blood diseases have a great use, iPS cells can treat nervous system diseases,to provide in vitro disease model, to study the mechanism of disease formation, screening new drugs and thedevelopment of new to provide a new treatment The The use of iPS cells as a nuclear donor cell, with the appropriatereceptor cells after fusion can be directly obtained transgenic animals. Not only can improve the genetic nature ofanimals, but also can break the boundaries of species and get the new animal traits that cannot achieve by usingtraditional mating methods. The research of iPS cells has been widely concerned, and it is the research hotspot in cellbiology and molecular biology. In this paper, the defi nition of iPS cells, the acquisition of iPS cells, the history ofdevelopment, the signifi cance of research, the progress of research, the application of iPS cells, and the problems ofiPS cells were reviewed.

scholarly journals Dysfunction telomeres in embryonic fibroblasts and cultured in vitro pluripotent stem cells of Rattus norvegicus (Rodentia, Muridae)

2019 ◽  
Vol 13 (3) ◽  
pp. 197-210
Author(s):  
Natalya S. Zhdanova ◽  
Evgenia A. Vaskova ◽  
Tatyana V. Karamysheva ◽  
Julia M. Minina ◽  
Nicolay Rubtsov ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Rattus Norvegicus ◽  
Embryonic Stem ◽  
Telomere Dysfunction ◽  
Embryonic Fibroblasts ◽  
Damage Response ◽  
Induced Pluripotent

We studied the level of spontaneous telomere dysfunction in Rattus norvegicus (Berkenhout, 1769) (Rodentia, Muridae) embryonic fibroblasts (rEFs) and in cultured in vitro rat pluripotent stem cells (rPSCs), embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs), on early passages and after prolonged cultivation. Among studied cell lines, rESCs showed the lowest level of telomere dysfunction, while the riPSCs demonstrated an elevated level on early passages of cultivation. In cultivation, the frequency of dysfunctional telomeres has increased in all studied cell lines; this is particularly true for dysfunctional telomeres occurring in G1 stage in riPSCs. The obtained data are mainly discussed in the connection with the specific structure of the telomere regions and their influence on the differential DNA damage response in them.

scholarly journals 191IN VIVO AND IN VITRO DIFFERENTIATION OF EMBRYONIC STEM CELLS DERIVED FROM PARTHENOGENETIC EMBRYOS IN MICE

2004 ◽  
Vol 16 (2) ◽  
pp. 217
Author(s):  
T. Mitani ◽  
T. Teramura ◽  
T. Tada ◽  
Y. Hosoi ◽  
A. Iritani
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Immunohistochemical Analysis ◽  
Embryoid Bodies ◽  
Germline Transmission ◽  
Feeder Layers ◽  
Parthenogenetic Embryos

Availability of embryonic stem (ES) cells opens the prospect for regenerative medicine. However, ES cells genetically mismatched to diseased individuals cause immunological rejection. In this study, we established ES cells from parthenogenetic embryos in mice and examined their pluripotency. Oocytes were collected from (C57BL/6xDBA)F1 mice (BDF1) by superovulation. Parthenogenetic diploid embryos were produced by activation treatment in 5mM SrCl2 in Ca2+-free KSOM medium for 2h, followed by cultivation in 5μgmL−1 cytochalasin B for 6h. The zonae pellucidae of embryos developed to the blastocyst stage in vitro were removed by a 5-min incubation in 0.5% pronase. Inner cell masses (ICMs) isolated immunosurgically were seeded on the feeder layers (mitomycin C-treated mouse embryonic fibroblasts) in DMEM supplemented with 15% Knock-Out Serum Replacement (Invitrogen), 2mM L-glutamine, non-essential amino acids, β-mercaptoethanol and 103UmL−1 of Leukemia inhibitory factor (LIF) at 37°C in a humidified atmosphere with 5% CO2 in air. The attached ICM cells were mechanically disaggregated and seeded on the fresh feeder layers. After several passages, parthenogenetic ES (PnES) cell lines were established. The efficacy of establishing PnES cell lines was 66% (37/56). To examine the characteristics of PnES cell lines, seven lines were subjected to histochemical and immunohistochemical analysis. All showed alkaline phosphatase activity and immunoreactivity to anti-SSEA-1 and anti-Oct4 antibodies. They maintained euploid sets of choromosomes at 29; 59%. PnES cells from two of the seven lines were injected into 59 host blastocysts obtained from ICR mice, resulting in 16 chimeric offspring (27%). In another experiment, injection of ICM cells and ES cells obtained from fertilized BDF1 blastocysts and ICM cells obtained from BDF1 parthenogenetic blastocysts also produced chimeric offspring (35%, 7/20; 46%, 6/13; and 53%, 10/19, respectively). However, no chimeric mouse with germline transmission was obtained from PnES cells. Injection of 1×107 of PnES cells into SCID mice formed teratocarcinomas. Immunohistochemical analysis showed cells positive for nestin (specific to neuroepitherial stem cells), Tu-J (class III β-tublin), NF-M (neurofilament), desmin (muscle), and albumin (hepatocytes), which indicated their differentiation potency to the cells derived from all three germ layers. Simple embryoid bodies produced from these cell lines were plated on tissue culture dishes under conditions for induction of differentiation. Immunohistochemistry and RT-PCR analysis showed their differentiation into neurons (NF-M, nestin), cardiomyocytes and hepato-like cells (albumin, α-fetoprotein). Our results indicate that PnES cells are pluripotent similar to the ES cells from fertilized embryos except for germline transmission and should be tested in cell replacement animal models.

Abstract 203: A Shh Coreceptor Cdo is Required for Efficient Cardiomyogenesis of Pluripotent Stem Cells

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Myong-Ho Jeong ◽  
Young-Eun Leem ◽  
Hyun-Ji Kim ◽  
Kyungjin Kang ◽  
Hana Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Heart Development ◽  
Connexin 43 ◽  
Troponin T ◽  
Es Cells ◽  
Embryonic Stem ◽  
Differentiation Potential ◽  
Shh Signaling

Sonic hedgehog (Shh) signaling plays an important role for early heart development, such as heart looping and cardiomyogenesis of pluripotent stem cells. A multifunctional receptor Cdo functions as a Shh coreceptor together with Boc and Gas1 to activate Shh signaling and these coreceptors seem to play compensatory roles in early heart development. Thus in this study, we examined the role of Cdo in cardiomyogenesis by utilizing an in vitro differentiation of pluripotent stem cells. Here we show that Cdo is required for efficient cardiomyogenesis of pluripotent stem cells by activation of Shh signaling. Cdo is induced concurrently with Shh signaling activation upon induction of cardiomyogenesis of P19 embryonal carcinoma (EC) cells. Cdo -depleted P19 EC and Cdo –/– mouse embryonic stem (ES) cells display decreased expression of key cardiac regulators, including Gata4, Nkx2.5 and Mef2c and this decrease coincides with reduced Shh signaling activities. Furthermore Cdo deficiency causes a stark reduction in formation of mature contractile cardiomyocytes. This defect in cardiomyogenesis is overcome by reactivation of Shh signaling at the early specification stage of cardiomyogenesis. The Shh agonist treatment restores differentiation capacities of Cdo -deficient ES cells into contractile cardiomyocytes by recovering both the expression of early cardiac regulators and structural genes such as cardiac troponin T and Connexin 43. Therefore Cdo is required for efficient cardiomyogenesis of pluripotent stem cells and an excellent target to improve the differentiation potential of stem cells for generation of transplantable cells to treat cardiomyopathies.

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