scholarly journals In vitro characterization of the human segmentation clock

2018 ◽  
Author(s):  
Margarete Diaz-Cuadros ◽  
Daniel E Wagner ◽  
Christoph Budjan ◽  
Alexis Hubaud ◽  
Jonathan Touboul ◽  
...  
Keyword(s):  
Es Cells ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Model Organisms ◽  
Segmentation Clock ◽  
Human Segmentation ◽  
Somite Formation ◽  
Induced Pluripotent

The vertebral column is characterized by the periodic arrangement of vertebrae along the anterior-posterior (AP) axis. This segmental or metameric organization is established early in embryogenesis when pairs of embryonic segments called somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock 1,2. While this oscillator has been well characterized in model organisms 1,2, whether a similar oscillator exists in humans remains unknown. We have previously shown that human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells can differentiate in vitro into PSM upon activation of the Wnt signaling pathway combined with BMP inhibition3. Here, we show that these human PSM cells exhibit Notch and YAP-dependent oscillations4 of the cyclic gene HES7 with a 5-hour period. Single cell RNA-sequencing comparison of the differentiating iPS cells with mouse PSM reveals that human PSM cells follow a similar differentiation path and exhibit a remarkably coordinated differentiation sequence. We also demonstrate that FGF signaling controls the phase and period of the oscillator. This contrasts with classical segmentation models such as the “Clock and Wavefront” 1,2,5, where FGF merely implements a signaling threshold specifying where oscillations stop. Overall, our work identifying the human segmentation clock represents an important breakthrough for human developmental biology.

scholarly journals Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Shixin Zhou ◽  
Yinan Liu ◽  
Ruopeng Feng ◽  
Caiyun Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neural Progenitor Cells ◽  
Es Cells ◽  
Ectopic Expression ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Global Gene Expression ◽  
Ips Cells ◽  
Reprogramming Efficiency ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of four Yamanaka factors. Here, we report that Survivin, an apoptosis inhibitor, can enhance iPS cells generation from human neural progenitor cells (NPCs) together with one factor OCT4 (1F-OCT4-Survivin). Compared with 1F-OCT4, Survivin accelerates the process of reprogramming from human NPCs. The neurocyte-originated induced pluripotent stem (NiPS) cells generated from 1F-OCT4-Survivin resemble human embryonic stem (hES) cells in morphology, surface markers, global gene expression profiling, and epigenetic status. Survivin keeps high expression in both iPS and ES cells. During the process of NiPS cell to neural cell differentiation, the expression of Survivin is rapidly decreased in protein level. The mechanism of Survivin promotion of reprogramming efficiency from NPCs may be associated with stabilization ofβ-catenin in WNT signaling pathway. This hypothesis is supported by experiments of RT-PCR, chromatin immune-precipitation, and Western blot in human ES cells. Our results showed overexpression of Survivin could improve the efficiency of reprogramming from NPCs to iPS cells by one factor OCT4 through stabilization of the key molecule,β-catenin.

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.

scholarly journals Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells

2021 ◽  
Author(s):  
Emi Sano ◽  
Ayaka Sakamoto ◽  
Natsumi Mimura ◽  
Ai Hirabayashi ◽  
Yukiko Muramoto ◽  
...  
Keyword(s):  
Individual Differences ◽  
Es Cells ◽  
Membrane Vesicles ◽  
Ips Cells ◽  
Genetic Differences ◽  
Progeny Virus ◽  
Double Membrane ◽  
Human Ips Cells ◽  
Induced Pluripotent

AbstractGenetic differences are a primary reason for differences in the susceptibility and severity of coronavirus disease 2019 (COVID-19). Because induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Notably, undifferentiated human iPS cells themselves cannot be infected bySARS-CoV-2. Using adenovirus vectors, here we found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected with SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, the budding of viral particles, the production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We also evaluated COVID-19 therapeutic drugs in ACE2-iPS cells and confirmed the strong antiviral effects of Remdesivir, EIDD-2801, and interferon-beta. In addition, we performed SARS-CoV-2 infection experiments on ACE2-iPS/ES cells from 8 individuals. Male iPS/ES cells were more capable of producing the virus as compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro, but they are also a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences.Graphical Abstract

scholarly journals Human ES and iPS Cells Display Less Drug Resistance Than Differentiated Cells, and Naïve-State Induction Further Decreases Drug Resistance

2020 ◽  
Author(s):  
Yulia Panina ◽  
Junko Yamane ◽  
Kenta Kobayashi ◽  
Hideko Sone ◽  
Wataru Fujibuchi
Keyword(s):  
Drug Resistance ◽  
Drug Exposure ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Ips Cells ◽  
Toxicity Assessment ◽  
Research Knowledge ◽  
Differentiated Cells ◽  
Induced Pluripotent

AbstractPluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

scholarly journals Stepwise in vitro induction of human somitic mesoderm and its derivatives

2020 ◽  
Author(s):  
Yoshihiro Yamanaka ◽  
Maya Uemura ◽  
Cantas Alev
Keyword(s):  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Developmental Process ◽  
Cell Types ◽  
Model Organisms ◽  
Human Segmentation ◽  
Induced Pluripotent ◽  
Somitic Mesoderm ◽  
In Vitro Induction

Abstract Our understanding of human somitogenesis is limited and largely based on insights gained from model organisms. Pluripotent stem cell-based in vitro approaches aiming to recapitulate distinct aspects of this core developmental process have recently been reported, including our recent paper on the in vitro recapitulation of the human segmentation clock1. Here we describe in detail our stepwise induction protocol of presomitic mesoderm (PSM), somitic mesoderm (SM), and its two major derivatives, sclerotome (SCL) and dermomyotome (DM) from human induced pluripotent stem cells (iPSCs). We further briefly address the subsequent molecular and functional analysis of these in vitro induced human mesodermal lineages and cell-types.

332 DERIVATION OF BOVINE-INDUCED PLURIPOTENT STEM CELLS BY piggyBac-MEDIATED REPROGRAMMING

2015 ◽  
Vol 27 (1) ◽  
pp. 255
Author(s):  
T. T. Rao ◽  
K. Dharmendra ◽  
G. Silke ◽  
W. Garrels ◽  
H. Niemann ◽  
...  
Keyword(s):  
Stem Cell ◽  
Insertional Mutagenesis ◽  
Bovine Genome ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Major Step ◽  
Livestock Species ◽  
Increased Risk ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells are a seminal breakthrough in stem cell research and are promising for the development of advanced regenerative therapies and farm animal biotechnology. Considering the potential of this technology for both basic and clinical research, it is tempting to extend this research to important livestock species, such as cattle, in which authentic embryonic stem cell lines are yet not available. 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 removable after reprogramming. Here, we describe a nonviral method for the derivation of bovine iPS cells, employing a piggyBac (PB) transposon system. The reprogramming PB transposon encodes the primate cDNA of 6 core reprogramming factors, OCT4, SOX2, KLF4, MYC, LIN28, and NANOG, separated by self-cleaving 2A peptide sequences and driven by the chimeric CAGGS promoter. The derived bovine iPS line expressed typical endogenous genes (OCT4, SOX2, c-MYC, KLF4, NANOG, REX1, and ALP) by RT-PCR and OCT-4 as well as SSEA-1 and 4 pluripotency-related markers by immunostaining, and it exhibited silencing of exogenous reprograming factors. Moreover, the iPS line showed long-term proliferation (until the 40th passage) under feeder-free culture conditions, differentiated into derivatives of the 3 germ layers in vitro, and formed teratomas (4/6) after subcutaneous injection into immunodeficient nude mice. These results are a major step towards the derivation of authentic bovine iPS cells, and thus facilitate the genetic modifications of the bovine genome.

Re-establishing pluripotency in adult cells derived from breast stromal tissue.

2011 ◽  
Vol 29 (27_suppl) ◽  
pp. 227-227
Author(s):  
S. M. L. Lim ◽  
I. Aksoy ◽  
K. G. C. Lim ◽  
J. Karuppasamy ◽  
U. Divakar ◽  
...  
Keyword(s):  
Cell Biology ◽  
Breast Tissue ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Dermal Fibroblasts ◽  
Patient Specific ◽  
Stromal Tissue ◽  
Induced Pluripotent

227 Background: Recent advances in pluripotent stem cell biology offer patient-specific disease models to investigate in vitro mechanisms of tumorigenesis. Induced pluripotent stem (iPS) cells were originally derived by reprogramming of human dermal fibroblasts through ectopic expression of pluripotency–associated transcription factors. A limitation to the use of dermal fibroblasts as the starting cell type for reprogramming is that it usually takes weeks to expand cells from a single biopsy, and the efficiency of the process is very low. In contrast, a large number of adipose-derived mesenchymal stromal cells (Ad-MSCs) can be easily obtained from the stroma of human breast tissue, without the time-consuming steps of cell expansion. Here we investigated the ability to induce pluripotency in committed, Ad-MSCs derived from the stroma of breast tissue. Methods: The aim of this study is to investigate the potential of using Ad-MSCs derived from surgically discarded breast stromal tissue to generate human iPS. Discarded tissue during surgical procedures was processed in vitro and Ad-MSCs were derived. These Ad-MSCs were then used to generate iPS cells by ectopic expression of “Yamanaka’s cocktail” containing OCT4, SOX2, KLF4 and c-MYC. Results: The success rate in generating iPS cells from human Ad-MSCs derived from breast stromal tissue is very high compared to the use of dermal fibroblasts. In our study, almost all human Ad-MSC cell lines can be reprogrammed into iPS cells, which share the same characteristics as skin fibroblast-derived iPS cells and human embryonic stem cells in their morphology, gene expression profile and differentiation capacities. Conclusions: We are now optimizing this approach and making it more clinically relevant by adopting an integration-free method to deliver the reprogramming factors. The successful reprogramming of breast stromal-derived Ad-MSCs into iPS cells may provide a valuable source of patient-specific iPS cells to study the mechanism of tumorigenesis in patients with breast cancer.

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