Porcine induced pluripotent stem cells require LIF and maintain their developmental potential in early stage of embryos.
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ABSTRACT:
SUBMITTER: Cheng D
PROVIDER: S-EPMC3522612 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.
| S-EPMC3190702 | biostudies-literature
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. Transcript profiling utilized Affymetrix porcine microarrays were conducted to compare the gene expressions associated with pluripotency in the two LIF-dependent pESK lines (pESK I & II, passage 14 & 5, respectively) with a naïve phenotype and two porcine iPSC lines (ID4 & ID6, both passage 7, GSE15472) with a primed phenotype that re-programming of porcine fetal fibroblasts (EGFP-PFF, GSE15472). The pESK lines clustered separately from the porcine iPSC lines, EGFP-PFF and primary cultures established from explants of porine umbilical cord (PUC).
2011-12-02 | GSE26369 | GEO
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. High throughput SNP chip genotyping was conducted on Illumina's Porcine SNP60 BeadChip (WG-410, a service provided by Geneseek, NE, http://www.neogen.com/GeneSeek/). The results exhibited that the two lines pluripotent stem cells from the inner cell mass of porcine blastocysts were porcine origin and genetically distinct.
2011-01-05 | GSE26436 | GEO
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naM-CM-/ve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. Transcript profiling utilized Affymetrix porcine microarrays were conducted to compare the gene expressions associated with pluripotency in the two LIF-dependent pESK lines (pESK I & II, passage 14 & 5, respectively) with a naM-CM-/ve phenotype and two porcine iPSC lines (ID4 & ID6, both passage 7, GSE15472) with a primed phenotype that re-programming of porcine fetal fibroblasts (EGFP-PFF, GSE15472). The pESK lines clustered separately from the porcine iPSC lines, EGFP-PFF and primary cultures established from explants of porine umbilical cord (PUC). Porcine pluripotent stem cells were derived from the inner cell mass with transduction with human KLF4 by lentiviral transduction.
2011-12-02 | E-GEOD-26369 | biostudies-arrayexpress
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. High throughput SNP chip genotyping was conducted on Illumina's Porcine SNP60 BeadChip (WG-410, a service provided by Geneseek, NE, http://www.neogen.com/GeneSeek/). The results exhibited that the two lines pluripotent stem cells from the inner cell mass of porcine blastocysts were porcine origin and genetically distinct. Porcine pluripotent stem cells were derived from the inner cell mass with transduction with human KLF4 by lentiviral transduction.
2011-01-05 | E-GEOD-26436 | biostudies-arrayexpress
Project description:Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.
| S-EPMC4798268 | biostudies-literature
Project description:PURPOSE: The aim of this study was to investigate the developmental potential of isolated blastomeres in the presence and absence of leukemia inhibitory factor (LIF) and granulocyte-macrophage colony stimulating factor (GM-CSF). METHODS: The blastomeres of two (1/2) and eight cells (1/8) embryos were isolated and cultured in T6 medium in the presence and absence of LIF (1,000 IU/ml) and or GM-CSF (2 ng/ml) up to 120 h. The diameter and cell number of blastocysts were measured. RESULTS: The developmental rates of 1/2 isolated blastomeres developed to blastocysts stages in the presence and absence of LIF and GM-CSF were 45.80, 35.10 and 48.66, 41.66, respectively. The diameter of blastocysts was higher in GM-CSF group and total cell number of blastocyst in both treated groups was higher than control (P < 0.05). No 1/8 blastomeres developed to morula and blastocyst stages. CONCLUSIONS: LIF and GM-CSF could improve the development of 1/2 isolated blastomeres.
| S-EPMC2582108 | biostudies-other
Project description:Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into ?-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established.
| S-EPMC4865852 | biostudies-other
Project description:BackgroundTo maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes.MethodsElectrospun nanofibrous substrates with different reduced Young's modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively.ResultsThe results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner.ConclusionsLineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications.
| S-EPMC5622562 | biostudies-literature
Project description:Endogenous cell signals regulate tissue homeostasis and are significant for directing the fate of stem cells. During liver development, cytokines released from various cell types are critical for stem/progenitor cell differentiation and lineage expansions. To determine mechanisms in these stage-specific lineage interactions, we modeled potential effects of soluble signals derived from immortalized human fetal liver parenchymal cells on stem cells, including embryonic and induced pluripotent stem cells. For identifying lineage conversion and maturation, we utilized conventional assays of cell morphology, gene expression analysis and lineage markers. Molecular pathway analysis used functional genomics approaches. Metabolic properties were analyzed to determine the extent of hepatic differentiation. Cell transplantation studies were performed in mice with drug-induced acute liver failure to elicit benefits in hepatic support and tissue regeneration. These studies showed signals emanating from fetal liver cells induced hepatic differentiation in stem cells. Gene expression profiling and comparison of regulatory networks in immature and mature hepatocytes revealed stem cell-derived hepatocytes represented early fetal-like stage. Unexpectedly, differentiation-inducing soluble signals constituted metabolomics products and not proteins. In stem cells exposed to signals from fetal cells, mechanistic gene networks of upstream regulators decreased pluripotency, while simultaneously inducing mesenchymal and epithelial properties. The extent of metabolic and synthetic functions in stem cell-derived hepatocytes was sufficient for providing hepatic support along with promotion of tissue repair to rescue mice in acute liver failure. During this rescue, paracrine factors from transplanted cells contributed in stimulating liver regeneration. We concluded that hepatic differentiation of pluripotent stem cells with metabolomics products will be significant for developing therapies. The differentiation mechanisms involving metabolomics products could have an impact on advancing recruitment of stem/progenitor cells during tissue homeostasis.
| S-EPMC6483088 | biostudies-literature