scholarly journals Establishment and Identification of a CiPSC Lineage Reprogrammed from FSP-tdTomato Mouse Embryonic Fibroblasts (MEFs)

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Ruiping Chen ◽  
Wenxiu Xie ◽  
Baomei Cai ◽  
Yue Qin ◽  
Chuman Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecule ◽  
Fluorescent Protein ◽  
Day Treatment ◽  
Tracking System ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Underlying Mechanisms

Safety issues associated with transcription factors or viruses may be avoided with the use of chemically induced pluripotent stem cells (CiPSCs), thus promoting their clinical application. Previously, we had successfully developed and standardized an induction method using small-molecule compound, with simple operation, uniform induction conditions, and clear constituents. In order to verify that the CiPSCs were indeed reprogrammed from mouse embryonic fibroblasts (MEFs), and further explore the underlying mechanisms, FSP-tdTomato mice were used to construct a fluorescent protein-tracking system of MEFs, for revealing the process of CiPSC reprogramming. CiPSCs were identified by morphological analysis, mRNA, and protein expression of pluripotency genes, as well as teratoma formation experiments. Results showed that after 40-day treatment of tdTomato-MEFs with small-molecule compounds, the cells were presented with prominent nucleoli, high core-to-cytoplasmic ratio, round shape, group and mass arrangement, and high expression of pluripotency gene. These cells could differentiate into three germ layer tissues in vivo. As indicated by the above results, tdTomato-MEFs could be reprogrammed into CiPSCs, a lineage that possesses pluripotency similar to mouse embryonic stem cells (mESCs), with the use of small-molecule compounds. The establishment of CiPSC lineage, tracked by fluorescent protein, would benefit further studies exploring its underlying mechanisms. With continuous expression of fluorescent proteins during cellular differentiation, this cell lineage could be used for tracking CiPSC transplantation and differentiation into functional cells.

scholarly journals Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0130332 ◽  
Author(s):  
Boxian Huang ◽  
Song Ning ◽  
Lili Zhuang ◽  
Chunyan Jiang ◽  
Yugui Cui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
The Self ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals The Cyclin-Dependent Kinase Inhibitor p21 Is a Crucial Target for Histone Deacetylase 1 as a Regulator of Cellular Proliferation

2009 ◽  
Vol 30 (5) ◽  
pp. 1171-1181 ◽  
Author(s):  
Gordin Zupkovitz ◽  
Reinhard Grausenburger ◽  
Reinhard Brunmeir ◽  
Silvia Senese ◽  
Julia Tischler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Hdac Inhibitors ◽  
Embryonic Stem ◽  
Cyclin Dependent Kinase ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Cyclin Dependent Kinase Inhibitor

ABSTRACT Histone deacetylases (HDACs) are chromatin-modifying enzymes that are involved in the regulation of proliferation, differentiation and development. HDAC inhibitors induce cell cycle arrest, differentiation, or apoptosis in tumor cells and are therefore promising antitumor agents. Numerous genes were found to be deregulated upon HDAC inhibitor treatment; however, the relevant target enzymes are still unidentified. HDAC1 is required for mouse development and unrestricted proliferation of embryonic stem cells. We show here that HDAC1 reversibly regulates cellular proliferation and represses the cyclin-dependent kinase inhibitor p21 in embryonic stem cells. Disruption of the p21 gene rescues the proliferation phenotype of HDAC1−/− embryonic stem cells but not the embryonic lethality of HDAC1−/− mice. In the absence of HDAC1, mouse embryonic fibroblasts scarcely undergo spontaneous immortalization and display increased p21 expression. Chromatin immunoprecipitation assays demonstrate a direct regulation of the p21 gene by HDAC1 in mouse embryonic fibroblasts. Transformation with simian virus 40 large T antigen or ablation of p21 restores normal immortalization of primary HDAC1−/− fibroblasts. Our data demonstrate that repression of the p21 gene is crucial for HDAC1-mediated control of proliferation and immortalization. HDAC1 might therefore be one of the relevant targets for HDAC inhibitors as anticancer drugs.

scholarly journals Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7

Cell Cycle ◽  
2016 ◽  
Vol 15 (21) ◽  
pp. 2931-2942 ◽  
Author(s):  
Areta M. Czerwinska ◽  
Joanna Nowacka ◽  
Magdalena Aszer ◽  
Sylwia Gawrzak ◽  
Karolina Archacka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Cycle Regulation ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Cycle Regulation

scholarly journals ID: 1021 Experimental reprogramming of murine embryonic fibroblasts towards induced pluripotent stem cells using a single polycistronic vector

2017 ◽  
Vol 4 (S) ◽  
pp. 96
Author(s):  
Oanh Thuy Huynh ◽  
Mai Thi-Hoang Truong ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Viral Vector ◽  
Embryonic Stem ◽  
Embryonic Fibroblasts ◽  
Induced Pluripotent ◽  
Teratoma Formation

Background: Embryonic stem cells are pluripotent, thus capable of differentiating into all types of cells derived from the three germ layers. However, the application of embryonic stem cells (ESCs) for preclinical and clinical studies is difficult due to ethical concerns. Induced pluripotent stem cells (iPSCs) are derived from differentiation and have many ESC characteristics. The study herein examines the production of iPSCs from reprogramming of mouse embryonic fibroblasts (MEFs) via transduction with defined factors.  Methods: MEFs were collected from mouse embryos via a previously published protocol. The cells were transduced with a single polycistronic viral vector encoding mouse cDNAs of Oct3/4, Sox2, Klf4 and c-Myc. Transduced cells were treated and sub- cultured with ESC medium. The cells were evaluated as iPSCs with specific morphology, and expression SSEA-1, Oct3/4, Sox2 and Nanog. In addition, they were also evaluated for pluripotency by assessing alkaline phosphatase (AP) activity and in vivo teratoma formation.  Results: Under the reprogrammed conditions, the transduced cells displayed a change in morphology, forming ESC-like clusters. These cell clusters strongly expressed pluripotent markers as well as ESC-specific genes. Furthermore, the colonies exhibited higher AP activity and formed teratomas when injected into the murine testis.  Conclusion: The study herein suggests that MEFs can be reprogrammed into iPSCs using a polycistronic viral vector encoding mouse cDNAs for Oct3/4, Sox2, Klf4 and c- Myc

scholarly journals Experimental reprogramming of murine embryonic fibroblasts towards induced pluripotent stem cells using a single polycistronic vector

2017 ◽  
Vol 4 (01) ◽  
pp. 159 ◽  
Author(s):  
Oanh Thuy Huynh ◽  
Mai Thi-Hoang Truong ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Viral Vector ◽  
Embryonic Stem ◽  
Embryonic Fibroblasts ◽  
Induced Pluripotent ◽  
Teratoma Formation

Introduction: Embryonic stem cells are pluripotent, thus capable of differentiating into all types of cells derived from the three germ layers. However, the application of embryonic stem cells (ESCs) for preclinical and clinical studies is difficult due to ethical concerns. Induced pluripotent stem cells (iPSCs) are derived from differentiation and have many ESC characteristics. The study herein examines the production of iPSCs from reprogramming of mouse embryonic fibroblasts (MEFs) via transduction with defined factors. Methods: MEFs were collected from mouse embryos via a previously published protocol. The cells were transduced with a single polycistronic viral vector encoding mouse cDNAs of Oct3/4, Sox2, Klf4 and c-Myc. Transduced cells were treated and sub-cultured with ESC medium. The cells were evaluated as iPSCs with specific morphology, and expression SSEA-1, Oct3/4, Sox2 and Nanog. In addition, they also were evaluated for pluripotency by assessing alkaline phosphatase (AP) activity and in vivo teratoma formation. Results: Under the reprogrammed conditions, the transduced cells displayed a change in morphology, forming ESC-like clusters. These cell clusters strongly expressed pluripotent markers as well as ESC-specific genes. Furthermore, the colonies exhibited higher AP activity and formed teratomas when injected into the murine testis. Conclusions: The study herein suggests that MEFs can be reprogrammed into iPSCs using a polycistronic viral vector encoding mouse cDNAs for Oct3/4, Sox2, Klf4 and c-Myc.  

231 Novel protocol for the invitro maintenance and expansion of adult epidermal LGR5+ stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 243
Author(s):  
N. Gupta ◽  
K. Polkoff ◽  
J. Piedrahita
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Fluorescent Protein ◽  
Basal Medium ◽  
Feeder Layer ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Donor Skin ◽  
Fetal Fibroblasts ◽  
Basal Media

Currently, the standard for treatment of full-thickness skin wounds is skin grafts or bioengineered skin substitutes; however, this method is limited by the amount of intact donor skin and lack of follicles and architecture. Thus, a protocol is needed for the expansion and differentiation of adult epidermal and hair follicle stem cells for use in scaffold mediated tissue engineering. Recently, we developed a transgenic porcine model in which H2B-GFP is under the control of the LGR5 promoter. LGR5 is an established marker of stem cells, meaning this model can be used to track the development and behaviour of these cells. The focus of this project was to create a novel culture method for the maintenance and expansion of LGR5+ epidermal adult stem cells utilising the green fluorescent protein (GFP) tag. Single cell epidermal stem cells were isolated from porcine skin using dispase II (10mgmL−1; Sigma) and trypsin (0.05%; Corning). Porcine fetal fibroblasts (PFF) or mouse embryonic fibroblasts (MEF) were grown to 95% confluence in a 6-well plate. Feeder layer cells were mitotically inactivated by incubation with mitomycin C (Sigma Aldrich, 10μgmL−1). Three different media were tested: basal medium [Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, Corning; Ham's F12, ThermoFisher; fetal bovine serum, Gemini Bio-Products], basal media + 5-azacytidine (Sigma Aldrich) and CHIR99021 (Tocris), or basal media + keratinocyte growth supplements (transferrin, hydrocortisone, T3, adenine, insulin, cholera toxin; Sigma Aldrich, epidermal growth factor; R&D Systems). Epidermal cells were plated in each medium for both PFF and MEF feeder layers. Experiments were performed in technical duplicates and replicated 3 times. On Day 9, total numbers of colonies in each well were counted and number of GFP-positive cells were quantified using ImageJ (National Institutes of Health). Results in Table 1 show that overall, the MEF feeder layer was able to support a higher rate of growth (P<0.05) and maintain the LGR5+ lineage at a higher proportion under all of the experimental conditions (P<0.05). In the growth-supplemented media, MEFs had fewer colonies than PFFs, but MEF colonies were, on average, 2.5 times larger (P<0.05). Conditions containing 5-aza and CHIR were the only conditions to maintain the LGR5+ lineage on the feeder layer. Statistically significant differences (P<0.05) were determined using two-way ANOVA, followed by Tukey's HSD test. Next, LGR5+ cells will be plated in media containing additional growth factors to stimulate expansion, while using CHIR and 5-aza to maintain the LGR5+ lineage. This protocol could be used in scaffolds to create three-dimensional growth of skin invitro and lead to better grafts for burn victims. Table 1.Growth of LGR5+ cells in different media including 5-azacytidine (5-aza), CHIR 99021 (CHIR), and keratinocyte growth supplements Group1 Basal medium (BM) BM + 5-aza+ CHIR BM + growth supplements No. of colonies/well MEF 127.7±40.8AB 189.3±16.9A 87.3±14.6B PFF 65.0±14.1A 83.3±17.0AB 148±33.7B Average no. of GFP+ cells per frame MEF 0.5±0.8B 65.7±18.4A 1.8±1.7B PFF 0.9±1.0B 22.6±4.5A 0.3±0.6B A,BValues within rows with different superscripts differ (P ≤ 0.05). 1MEF=mouse embryonic fibroblasts; PFF=porcine fetal fibroblasts.

Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4598-4603 ◽  
Author(s):  
Lisheng Wang ◽  
Li Li ◽  
Pablo Menendez ◽  
Chantal Cerdan ◽  
Mickie Bhatia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Conditioned Media ◽  
Proliferative Potential ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Self Renewal ◽  
Hematopoietic Development

Abstract To date, hematopoietic development of human embryonic stem cells (hESCs) has been limited to cell lines cultured in the presence of either mouse embryonic fibroblasts (MEFs) or MEF-conditioned media (MEF-CM). Anonymous xenogenic factors from MEFs or MEF-CM complicate studies of hESC self-renewal and also raise concerns for the potential clinical applications of generating primitive hematopoietic cells from hESC lines maintained under these ambiguous conditions. Here, we demonstrate that hESCs can be cultured over 30 passages in defined conditions in the absence of MEFs or MEF-CM using only serum replacement (SR) media and high concentrations of basic fibroblast growth factor (SR-bFGF). Similar to hESCs cultured in MEF-CM, hESCs cultured in SR-bFGF sustained characteristics of undifferentiated hESCs, proliferative potential, normal karyotype, in vitro and in vivo 3 germ-layer specification and gave rise to hemogenic-endothelial precursors required for subsequent primitive hematopoietic development. Our report demonstrates that anonymous factors produced by feeder cells are not necessary for hESC maintenance and subsequent hematopoietic specification, thereby providing a defined system for studies of hESC self-renewal and hESC-derived hematopoiesis. (Blood. 2005;105:4598-4603)

scholarly journals Induction of Pluripotent Stem Cells from Mouse Embryonic Fibroblasts by Oct4 and Klf4 with Small-Molecule Compounds

2008 ◽  
Vol 3 (5) ◽  
pp. 568-574 ◽  
Author(s):  
Yan Shi ◽  
Caroline Desponts ◽  
Jeong Tae Do ◽  
Heung Sik Hahm ◽  
Hans R. Schöler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts
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