LCDM medium supports the derivation of bovine extended pluripotent stem cells with embryonic and extraembryonic potency in bovine‐mouse chimeras from iPSCs and bovine fetal fibroblasts

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

Download Full-text

Derivation of induced pluripotent stem cells from ferret somatic cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00456.2019 ◽

2020 ◽

Vol 318 (4) ◽

pp. L671-L683

Author(s):

Jinghui Gao ◽

Sophia Petraki ◽

Xingshen Sun ◽

Leonard A. Brooks ◽

Thomas J. Lynch ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Preclinical Model ◽

Specific Cell ◽

Chronic Lung Diseases ◽

Human Ipscs ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.

Download Full-text

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.709286 ◽

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.

Download Full-text

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions

Research in Veterinary Science ◽

10.1016/j.rvsc.2019.01.015 ◽

2019 ◽

Vol 123 ◽

pp. 252-260 ◽

Cited By ~ 2

Author(s):

Deepak Kumar ◽

Taruna Anand ◽

Kennady Vijayalakshmy ◽

Papori Sharma ◽

Rasika Rajendran ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Culture Conditions ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Download Full-text

222 INCOMPLETE REPROGRAMMING OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PORCINE FETAL FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab222 ◽

2016 ◽

Vol 28 (2) ◽

pp. 242

Author(s):

K.-H. Choi ◽

D. Son ◽

D.-K. Lee ◽

J.-N. Oh ◽

S.-H. Kim ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Ectopic Expression ◽

Cellular Reprogramming ◽

Pluripotent State ◽

Fetal Fibroblasts ◽

Endogenous Genes ◽

Induced Pluripotent

Cellular reprogramming of committed cells into a pluripotent state can be accomplished by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. However, during reprogramming, it has been verified that failures of reactivating endogenous genes and epigenetic remodelling lead to partially reprogrammed cells exhibiting features similar to those of fully reprogrammed cells. In this study, partially reprogrammed induced pluripotent stem cells (pre-iPSC) were derived from porcine fetal fibroblasts via drug-inducible vector carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study aimed to investigate characteristics of pre-iPSC and reprogramming mechanisms. The pre-iPSC were stably maintained over an extended period having in vitro differentiation ability into 3 germ layers. The pluripotent state of pre-iPSC was regulated by modulation of culture condition. They showed naive- or primed-like pluripotent state in leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) supplemented culture conditions respectively. However, pre-iPSC could not be maintained without ectopic expression of transgenes. The cultured pre-iPSC expressed endogenous transcription factors (OCT4 and SOX2) except for NANOG known as gateway into complete reprogramming. In addition, endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occurred in female pre-iPSC. Given the results, we found that expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu for achieving faithful pluripotency in pig. Accordingly, investigating pre-iPSC could help us to improve and develop reprogramming methods via understanding reprogramming mechanisms in pig. This work was supported by the Next-generation BioGreen 21 Program (PJ0113002015), Rural Development Administration, Republic of Korea.

Download Full-text

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts

Molecular Medicine Reports ◽

10.3892/mmr.2017.6431 ◽

2017 ◽

Vol 15 (6) ◽

pp. 3690-3698 ◽

Cited By ~ 5

Author(s):

Yuehong Wu ◽

Ouyang Li ◽

Chengwen He ◽

Yong Li ◽

Min Li ◽

...

Keyword(s):

Stem Cells ◽

Guinea Pig ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Download Full-text

Generation of Induced Pluripotent Stem Cells From Buffalo (Bubalus bubalis) Fetal Fibroblasts with Buffalo Defined Factors

Stem Cells and Development ◽

10.1089/scd.2012.0018 ◽

2012 ◽

Vol 21 (13) ◽

pp. 2485-2494 ◽

Cited By ~ 29

Author(s):

Yanfei Deng ◽

Qingyou Liu ◽

Chan Luo ◽

Shibei Chen ◽

Xiangping Li ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Bubalus Bubalis ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Download Full-text

Improved Efficiency and Pace of Generating Induced Pluripotent Stem Cells from Human Adult and Fetal Fibroblasts

Stem Cells ◽

10.1634/stemcells.2008-0346 ◽

2008 ◽

Vol 26 (8) ◽

pp. 1998-2005 ◽

Cited By ~ 202

Author(s):

Prashant Mali ◽

Zhaohui Ye ◽

Holly H. Hommond ◽

Xiaobing Yu ◽

Jeff Lin ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Human Adult ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Download Full-text

Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules

10.1101/2020.05.21.107862 ◽

2020 ◽

Author(s):

Stoyan Petkov ◽

Ralf Dressel ◽

Ignacio Rodriguez-Polo ◽

Rüdiger Behr

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Model ◽

Common Marmoset ◽

Animal Disease ◽

Neural Lineage ◽

Somatic Cell Reprogramming ◽

Fetal Fibroblasts

SUMMARYInduced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies for many currently incurable diseases. However, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Moreover, in order to reduce possible risks for the patients, it is necessary to use reprogramming approaches that ensure to the greatest extent possible the genomic integrity of the cells. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fetal fibroblasts with Tomato-modified VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC (VEE-OKS-iM-iTomato) and culture in medium supplemented with two small molecule inhibitors, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. The resulting cell lines possess pluripotency characteristics, such as expression of various pluripotency markers, long-term self-renewal, stable karyotype, and ability to differentiate into derivatives of the three primary germ layers in vitro and in vivo. Our experiments reveal a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either towards the neural lineage or further reprogrammed to pluripotency. These results (i) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (ii) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

Download Full-text