scholarly journals Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species

Development ◽  
2021 ◽  
Vol 148 (23) ◽  
Author(s):  
Masaki Kinoshita ◽  
Toshihiro Kobayashi ◽  
Benjamin Planells ◽  
Doris Klisch ◽  
Daniel Spindlow ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Genetic Manipulation ◽  
Self Renewal ◽  
Serum Factors ◽  
Global Comparison ◽  
Embryonic Disc ◽  
Wnt Inhibitor ◽  
Defined Culture

ABSTRACT Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated ‘The people behind the papers’ interview.

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).

scholarly journals Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Warunya Chakritbudsabong ◽  
Somjit Chaiwattanarungruengpaisan ◽  
Ladawan Sariya ◽  
Sirikron Pamonsupornvichit ◽  
Joao N. Ferreira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

scholarly journals Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

2019 ◽  
Author(s):  
Richard L Carpenedo ◽  
Sarah Y Kwon ◽  
R Matthew Tanner ◽  
Julien Yockell-Lelièvre ◽  
Chandarong Choey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Biology ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Endoderm Differentiation ◽  
Prolonged Differentiation ◽  
Defined Culture

SummaryHuman pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

scholarly journals Ascorbic Acid Can Promote the Generation and Expansion of Neuroepithelial-Like Stem Cells Derived from HiPS/ES Cells Under Chemically Defined Conditions Through Promoting Collagen Synthesis

2020 ◽  
Author(s):  
Rui Bai ◽  
Yun Chang ◽  
Amina Saleem ◽  
Fujian Wu ◽  
Lei Tian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ascorbic Acid ◽  
Pluripotent Stem Cells ◽  
Culture System ◽  
Serum Free Medium ◽  
Cord Injury ◽  
Induction System ◽  
Defined Culture

Abstract Introduction: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs) which hold promise in therapy for SCI by transplantation. In our study, we aimed to establish a chemically defined culture system by using serum-free medium and ascorbic acid (AA) to generation and expansion of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods: We induce hESC/iPSC to neurospheres by using a newly established induction system in vitro in our study. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction systems, including conventional N2 medium with gelatin-coated (coated) and N2+AA medium without pre-coated (AA) were characterized by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons and the microelectrode array (MEA) recording system was used to evaluate the functionality of neurons differentiated from lt-NES cells. Moreover, the mechanism of AA-induced lt-NES cells was explored through RNA-seq and the use of inhibitors. Results: HESCs/iPSCs were efficiently induced to neurospheres by using a newly established induction system in vitro. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction system (coated vs AA) both expressed neural pluripotency-associated genes PAX6, NESTIN, SOX1, SOX2. After long-term cultivation, we found that they both can maintain the long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retain the ability to differentiate into general functional neurons that highly express β-tubulin. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathways. Conclusions: Taken together, this new chemically defined culture system is stable and effective to generate and culture the lt-NES cells induced by hESCs/iPSCs using serum-free medium combined with ascorbic acid (AA). The lt-NES cells under this culture system can maintain the long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Keywords: Spinal cord injury, Neurospheres, Ascorbic acid, lt-NES cells, Human pluripotent stem cells.

scholarly journals Genome maintenance in pluripotent stem cells

2014 ◽  
Vol 204 (2) ◽  
pp. 153-163 ◽  
Author(s):  
Uri Weissbein ◽  
Nissim Benvenisty ◽  
Uri Ben-David
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
In Vitro Propagation ◽  
Basic Research ◽  
Culture Conditions ◽  
Genome Maintenance ◽  
Self Renewal ◽  
Genomic Aberrations

Pluripotent stem cells (PSCs) must maintain their proper genomic content in order to preserve appropriate self-renewal and differentiation capacities. However, their prolonged in vitro propagation, as well as the environmental culture conditions, present serious challenges to genome maintenance. Recent work has been focused on potential means to alleviate the genomic insults experienced by PSCs, and to detect them as soon as they arise, in order to prevent the detrimental consequences of these genomic aberrations on PSC application in basic research and regenerative medicine.

scholarly journals An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

2017 ◽  
Author(s):  
Jianying Guo ◽  
Dacheng Ma ◽  
Rujin Huang ◽  
Jia Ming ◽  
Min Ye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Genetic Manipulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Human Pluripotent Stem Cells ◽  
Hesc Line ◽  
Transcription Activator

AbstractHuman pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level did not only promote naïve pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

scholarly journals Defined Culture Conditions Accelerate Small-molecule-assisted Neural Induction for the Production of Neural Progenitors from Human-induced Pluripotent Stem Cells

2017 ◽  
Vol 26 (12) ◽  
pp. 1890-1902 ◽  
Author(s):  
Patrick Walsh ◽  
Vincent Truong ◽  
Caitlin Hill ◽  
Nicolas D. Stoflet ◽  
Jessica Baden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Early Stage ◽  
Neural Induction ◽  
Immunocompromised Host ◽  
Neural Progenitors ◽  
Induced Pluripotent ◽  
Defined Culture

The use of defined conditions for derivation, maintenance, and differentiation of human-induced pluripotent stem cells (hiPSCs) provides a superior experimental platform to discover culture responses to differentiation cues and elucidate the basic requirements for cell differentiation and fate restriction. Adoption of defined systems for reprogramming, undifferentiated growth, and differentiation of hiPSCs was found to significantly influence early stage differentiation signaling requirements and temporal kinetics for the production of primitive neuroectoderm. The bone morphogenic protein receptor agonist LDN-193189 was found to be necessary and sufficient for neural induction in a monolayer system with landmark antigens paired box 6 and sex-determining region Y-box 1 appearing within 72 h. Preliminary evidence suggests this neuroepithelium was further differentiated to generate ventral spinal neural progenitors that produced electrophysiologically active neurons in vitro, maintaining viability posttransplantation in an immunocompromised host. Our findings support current developments in the field, demonstrating that adoption of defined reagents for the culture and manipulation of pluripotent stem cells is advantages in terms of simplification and acceleration of differentiation protocols, which will be critical for future clinical translation.

scholarly journals Derivation of sheep embryonic stem cells under optimized conditions

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 761-772 ◽  
Author(s):  
Marcela Vilarino ◽  
Delia Alba Soto ◽  
Yanina Soledad Bogliotti ◽  
Leqian Yu ◽  
Yanli Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
De Novo ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Livestock Species ◽  
Agricultural Applications ◽  
Wnt Inhibitor ◽  
Defined Culture

Until recently, it has been difficult to derive and maintain stable embryonic stem cells lines from livestock species. Sheep ESCs with characteristics similar to those described for rodents and primates have not been produced. We report the derivation of sheep ESCs under a chemically defined culture system containing fibroblast growth factor 2 (FGF2) and a tankyrase/Wnt inhibitor (IWR1). We also show that several culture conditions used for stabilizing naïve and intermediate pluripotency states in humans and mice were unsuitable to maintain ovine pluripotency in vitro. Sheep ESCs display a smooth dome-shaped colony morphology, and maintain an euploid karyotype and stable expression of pluripotency markers after more than 40 passages. We further demonstrate that IWR1 and FGF2 are essential for the maintenance of an undifferentiated state in de novo derived sheep ESCs. The derivation of stable pluripotent cell lines from sheep blastocysts represents a step forward toward understanding pluripotency regulation in livestock species and developing novel biomedical and agricultural applications.

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.

scholarly journals Pluripotent stem cells: induction and self-renewal

2018 ◽  
Vol 373 (1750) ◽  
pp. 20170213 ◽  
Author(s):  
R. Abu-Dawud ◽  
N. Graffmann ◽  
S. Ferber ◽  
W. Wruck ◽  
J. Adjaye
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Self Renewal

Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers—mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, good manufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state. We propose these as additional biomarkers defining naive PSCs. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

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