330 DERIVATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS FROM FIBROBLASTS OF A TRANSLOCATED AZOOSPERMIC BOAR

2015 ◽  
Vol 27 (1) ◽  
pp. 254
Author(s):  
A. Congras ◽  
H. Barasc ◽  
C. Delcros ◽  
F. Vignoles ◽  
A. Pinton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Lines ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Genomic Stability ◽  
Comparative Genomic ◽  
Differentiation Potential ◽  
Ips Cell ◽  
The Impact

Chromosomal rearrangements have a crucial impact on the proper proceedings of meiosis and can lead by several mechanisms to the production of unbalanced gametes or to the complete arrest of gametes production. To assess the impact of these rearrangements in the early development of pig germ cells, we proposed to generate a library of stem cells from infertile boars that are carriers of chromosomal abnormalities as a new tool for the development of an in vitro differentiation system from pluripotent stem cells to germ cells. We report here the reprogramming of fibroblasts from an azoospermic boar carrying a reciprocal translocation t(Y:14) by integrative or nonintegrative viral overexpression of Oct4, Sox2, Klf4, and c-Myc. The iPS cell lines were characterised for pluripotency, cell cycle, and differentiation potential by conventional methods. Genomic stability was analysed by G-banding karyotype, comparative genomic hybridization, and FISH. The porcine iPS-like cell lines harbored characteristics of ground and naïve pluripotency when cultured in specific media. They expressed several pluripotency genes and harbored an ES-like cell cycle. Nevertheless, contrary to mouse and human iPS, they did not silence the integrated exogenes, leading to a poor differentiation potential. Moreover, cytogenetic analysis revealed a high genomic instability upon passaging, which suggests the development of a population with an increased selective advantage. We characterised the selected duplications and compared them to those previously described in other species. In contrast, the nonintegrative reprogrammation system gives us promising results regarding differentiation potential and genomic stability and will bring new insights into the molecular factors controlling and maintaining pluripotency in the pig species.

scholarly journals A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

2015 ◽  
Vol 210 (7) ◽  
pp. 1257-1268 ◽  
Author(s):  
Sundari Chetty ◽  
Elise N. Engquist ◽  
Elie Mehanna ◽  
Kathy O. Lui ◽  
Alexander M. Tsankov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Positive Effects ◽  
Src Inhibitor ◽  
Expression Of Genes

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.

scholarly journals Long-Term Stability and Differentiation Potential of Cryopreserved cGMP-Compliant Human Induced Pluripotent Stem Cells

2019 ◽  
Vol 21 (1) ◽  
pp. 108 ◽  
Author(s):  
Mehdi Shafa ◽  
Tylor Walsh ◽  
Krishna Morgan Panchalingam ◽  
Thomas Richardson ◽  
Laura Menendez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Genomic Stability ◽  
Directed Differentiation ◽  
Differentiation Potential ◽  
Long Term Stability ◽  
Induced Pluripotent ◽  
Cell Banks ◽  
2D And 3D

The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products.

scholarly journals Using induced pluripotent stem cells to investigate human neuronal phenotypes in 1q21.1 deletion and duplication syndrome

2021 ◽  
Author(s):  
Gareth Chapman ◽  
Mouhamed Alsaqati ◽  
Sharna Lunn ◽  
Tanya Singh ◽  
Stefanie C Linden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Motor Deficits ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
The Impact ◽  
1Q21.1 Deletion

AbstractCopy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density, and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

scholarly journals 178 ESTABLISHMENT OF MOUSE PLURIPOTENT STEM CELLS GENERATED FROM PRIMORDIAL GERM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 239
Author(s):  
S.W. Shim ◽  
S.J. Song ◽  
H.S. Shim ◽  
S.J. Uhm ◽  
B.Y. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Cultured Cells ◽  
Cell Layer ◽  
Es Cells ◽  
Primordial Germ Cells ◽  
Pcr Analysis ◽  
Feeder Cell

Pluripotent stem cells have been generated from two embryonic sources: ES cells generated from ICM of blastocyst stage embryos, and embryonic germ (EG) cells generated from primordial germ cells (PGCs). Both ES and EG cells are pluripotent and exhibit important characteristics such as high alkaline phosphatase (AP) activity, multicellular colony formation, normal and stable karyotype, continuous passaging ability, and capacity to differentiate into three embryonic germ layers. This study was performed to establish the culture system for mouse EG cells derived from mouse PGCs. PGCs collected from the genital ridge of Day 11.5, 12.5, and 13.5 mouse embryos (C57BL/6 × DBA/2) were cultured and subsequently passaged on mitotically inactivated STO feeder cell layer. Cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, 1,000 units/mL of leukemia inhibiting factor (LIF), 6 ng/mL of SCF, and 10 ng/mL of bFGF at culture conditions of 5% CO2 in air, 95% relative humidity, 37°C temperature. Cells were routinely passaged every 3–4 days. Over a period of 7–10 days in primary culture, PGCs proliferated to form small, densely packed, multicellular colonies consisting of AP-positive cells that morphologically resembled undifferentiated ES cells. RT-PCR analysis confirmed mRNA expression of transcription factors Oct-4 and Nanog in these cells. Cultured cells could be maintained on the feeder cell layer for at least 10 passages and still retain normal karyotype. These results suggest that cell lines derived from mouse primordial germ cells are presumably EG cell lines and could be useful for transgenic animal production and ES cell study.

scholarly journals Potential Threats of Nanoplastic Accumulation in Human Induced Pluripotent Stem Cells

2021 ◽  
Author(s):  
Hyejoong Jeong ◽  
Wijin Kim ◽  
Daheui Choi ◽  
Jiwoong Heo ◽  
Uiyoung Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cellular Functions ◽  
Differentiation Potential ◽  
Developmental Toxicology ◽  
Neural Lineage ◽  
Induced Pluripotent ◽  
The Impact

Abstract Background: Micro- and nanoplastics (NPs) produced from the bulk fragmentation are rampant in the world by enormous plastic trash everyday life. NPs can be exposed to humans through a variety of routes, including inhalation and intake. The threat to humans from NPs is increasing invisibly. Nowadays, many people are concerned about human safety and health, but few are reported about the effects of NP on humans. To overcome the limitations in human studies, human induced pluripotent stem cells (hiPSCs) were used as an optimal in vitro platform to investigate developmental toxicology and subtle changes on cellular functions in terms of differentiation potential throughout a long-term culture. Results: Negatively charged polystyrene nanoplastics (PS NPs) were used to exclude acute toxic issues of surface charge and investigate the impact of the NP's size and nature during bioaccumulation. Intracellular observations revealed that NPs up to 1000 nm were over-internalized into single cells within 48 h, and smaller NPs demonstrated greater potency at decreasing number of viable cells by a strong correlation with the number of NPs on an equal mass basis. Also, PS NPs caused a significant reduction in self-renewal capacity of hiPSCs for 48 h. After the cells were exposed to PS NPs for 48 to 96 h at the beginning of the differentiation process, NPs accumulated in hiPSC did not render cellular functions vulnerable or adversely affect EB formation, EB-mediated differentiation, and neural lineage differentiation for up to 14 days.Conclusion: This study confirmed that hiPSC exposure to polystyrene nanoplastics results in acute toxicity and non-significant long-term effects on cellular functions. This report is important for understanding the developmental toxicology of nanoplastics and the origin of disease.

scholarly journals Oocyte Arrested at Metaphase II Stage Were Derived From Human Pluripotent Stem Cells in Vitro

2021 ◽  
Author(s):  
Xiaoli Yu ◽  
Ning Wang ◽  
Yingxin Zhang ◽  
Xiang Wang ◽  
Yikai Qiu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Polar Body ◽  
Primordial Follicle ◽  
Preimplantation Embryos ◽  
Differentiation Potential ◽  
Cell Medium

Abstract BackgroundGeneration and maturation of human oocyte in vitro could facilitate studies of folliculogenesis and oogenesis. We have previously shown that human aminotic fluid stem cells giving rise to oocyte-like cells (OLCs), However, it was difficult to observe whether these OLCs enter meiotic stage. MethodsHuman induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured by follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Surface marker expression and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing.ResultsTo induce hiPSCs differentiation into OLCs, cells were firstly cultured in a primordial germ cell medium for 10 days. The cells showed the morphology similar to primordial germ cells (PGCs), highly expressing germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured in the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cumulus-oocyte-complexes (COC) structures and OLCs in different sizes (50-150 μm diameter) with zona pellucida were observed. The in vitro matured OLCs presented the polar body and arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet proved.ConclusionsIn vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.

Impacts of microRNA-215 on cell proliferation and chemotherapy resistance in colon cancer and osteosarcoma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 2542-2542
Author(s):  
J. Ju ◽  
B. Song ◽  
Y. Wang
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Colon Cancer ◽  
Cell Proliferation ◽  
Cancer Stem Cells ◽  
Cell Lines ◽  
Chemotherapy Resistance ◽  
Osteosarcoma Cell ◽  
Colon Cancer Stem Cells ◽  
The Impact

2542 Background: Translational control plays a key role in resistance to anti-cancer drug treatment. MicroRNAs regulate gene expression at the post-transcriptional level, mainly by interacting with 3'-UTR of their mRNA targets. Methods: miR-215 was ectopically expressed by transient transfection in both human colon cancer cell lines and osteosarcoma cell lines. The impact of miR-215 on cell proliferation, cell cycle control, chemosensitivity and down stream targets were characterized. The expression of miR-215 in colorectal cancer specimens and normal adjacent tissues was quantified by real time-qRT-PCR analysis. Results: In this study, we discovered that miR-215 down-regulates the expression of both dihydrofolate reductase (DHFR) and thymidylate synthase (TS), two of the most important chemotherapeutic targets, in human osteosarcoma U-2 OS and colon cancer HCT-116 (wt-p53) cell lines. Cells with elevated miR-215 expression are more resistant to DHFR inhibitor methotrexate (MTX) or TS inhibitor Tomudex (TDX) treatment. Ectopically over-expressing miR-215 triggers reduced cell proliferation and increased G2 arrest, at least in part, through the induction of p53 and p21. miR-215 transfected cells with reduced proliferating phenotype were resist to MTX or TDX treatment due to deceased cell cycle in S phase. The expression of endogeneous miR-215 was highly elevated in CD133+/HI CD44+/HI colon cancer stem cells compared to CD133- CD44- colon cancer cells, suggesting that tumor stem cells may be avoiding cellular and DNA damage caused by chemotherapy with a reduced proliferating phenotype mediated by certain miRNAs such as miR-215. The elevated expression of miR-215 in colon cancer stem cells with slow proliferation rate and resistance to chemotherapy further supports the role of miR-215 in cell proliferation and chemotherapy resistance. Conclusions: miR-215 may have a unique potential as a novel therapeutic target and biomarker candidate in cancer. No significant financial relationships to disclose.

scholarly journals Using induced pluripotent stem cells to investigate human neuronal phenotypes in 1q21.1 deletion and duplication syndrome

2021 ◽  
Author(s):  
Gareth Chapman ◽  
Mouhamed Alsaqati ◽  
Sharna Lunn ◽  
Tanya Singh ◽  
Stefanie C. Linden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Motor Deficits ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
The Impact ◽  
1Q21.1 Deletion

AbstractCopy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

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