216 Oxygen levels and pluripotency maintenance supplements affect cellular reprogramming of bovine fibroblasts

2020 ◽  
Vol 32 (2) ◽  
pp. 236
Author(s):  
B. Bessi ◽  
R. Botigelli ◽  
K. Recchia ◽  
N. Pieri ◽  
G. Barbosa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Mek Inhibitor ◽  
Cellular Reprogramming ◽  
Embryoid Bodies ◽  
Experimental Conditions ◽  
Pluripotency Gene ◽  
Oxygen Tensions ◽  
Induced Pluripotent ◽  
Pluripotency Maintenance

Different supplements are used during invitro cellular reprogramming, usually acting on pluripotency maintenance and/or differentiation inhibition, such as basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and 2i (MEK inhibitor: PD0325901 + GSK3 inhibitor: CHIR99021). Another important factor affecting the reprogramming process is the oxygen (O2) tension because O2 levels can modify cellular metabolism and epigenetic markers, which are known to modulate pluripotency. Our objective was to evaluate the efficiency of reprogramming bovine fibroblasts in combination with different oxygen tensions (high O2, hO2×low O2, lO2) in different cell differentiation inhibitors: bFGF and bFGF + LIF + 2i (FL2i). Bovine fibroblasts were transduced with lentivirus harbouring mouse OSKM transcription factors (OCT4, SOX2, KLF4, and cMYC). Three clonal lineages were analysed for each experimental group. Pluripotency was characterised by morphology, detection of alkaline phosphatase, formation of embryoid bodies, and analysis of gene expression. As an initial pluripotency test, all colonies were positive for alkaline phosphatase (AP) activity in passages 5 to 6. Colonies were cultured for at least 15 passages (±140 days) with the exception of bFL2i colonies cultured in lO2, which did not grow beyond 7 to 8 passages. For gene expression analysis, samples of each colony in passages 5, 10, and 15 were used. When gene expression was analysed, both endogenous NANOG and OCT4 were increased in the bFGF group when cultured in hO2, and bFGF cultured in lO2 was higher than in the FL2i group (P<0.05). Also, NANOG was increased in early passages compared with late passages (P<0.05); SOX2 and FGF5 were increased in lO2 groups (P<0.05). The bFGF treatment increased STELLA expression compared with bFL2i (P<0.05) at both oxygen tensions. Interestingly, exogenous vector expression increased in the bFGF group compared with bFL2i (P<0.05) but was not affected by oxygen tension (P>0.05). All colonies tested were able to form embryoid bodies. In conclusion, it was not possible to maintain bovine induced pluripotent stem cells (biPS) in bFL2i treatment cultured in lO2 because these colonies were not able to remain viable after 8 passages. Moreover, small molecule supplementation strongly affected pluripotency gene expression. Further analysis on epigenetic changes, metabolism, and self-renewal is necessary to understand the pluripotent state in biPS under our experimental conditions. We acknowledge FAPESP for funding (grant 2015/26816-5 and fellowship 2018/24520-7).

scholarly journals Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1531
Author(s):  
Brendon Willian Bessi ◽  
Ramon Cesar Botigelli ◽  
Naira Caroline Godoy Pieri ◽  
Lucas Simões Machado ◽  
Jessica Brunhara Cruz ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Culture Conditions ◽  
Cellular Reprogramming ◽  
Embryoid Bodies ◽  
Low Oxygen ◽  
Loss Of Self ◽  
Clonal Lines ◽  
Induced Pluripotent ◽  
Pluripotency Maintenance

The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i—bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

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.

214 Different pluripotency maintenance supplements affect the reprogramming process and pluripotency state of bovine-induced pluripotent stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 235
Author(s):  
R. Botigelli ◽  
N. Pieri ◽  
B. Bessi ◽  
R. de Castro ◽  
K. Recchia ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Cell Reprogramming ◽  
Rt Pcr ◽  
Serum Replacement ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement ◽  
Pluripotency Maintenance

After the emergence of induced cell reprogramming, achieved through the addition of Yamanaka transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) to somatic cells, the number of studies regarding induction and maintenance of pluripotency has increased greatly. The success of bovine iPSCs (biPSCs) was first described by Summer et al. (2011 J. Anim. Sci. 89, 2708-2716; https://doi.org/10.2527/jas.2010-3666); however, investigations on the pluripotent state of biPSCs are still needed because different protocols and characterisation profiles have since been used. The aim of this study was to produce biPSC lines supplemented with different pluripotency maintenance agents to improve self-renewal and pluripotency maintenance. For that, bovine fetal (50 days) fibroblasts (3×104) were transduced with lentivirus harbouring mouse OSKM transcription factors. The cells were further cultured in reprogramming medium (Dulbecco's modified Eagle's medium/F12 KO and 20% KSR (knockout serum replacement)) supplemented with basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), bFGF+2i or LIF+2i (where 2i inhibitors are PD0325901 and CHIR99021). The capacity for cell reprogramming was analysed by colony formation and maintenance after manually and enzymatic passaging and alkaline phosphatase (AP) activity detection; additionally, pluripotency state was assessed by reverse transcription (RT)-PCR (pluripotency biomarkers: OCT4, NANOG, and SOX2; naïve state: STELLA, LIFr, and ESRRb; primed state: OTX2 and FGF5; and mouse (m)OSKM and invitro differentiation assay (embryoid body formation). Statistical analysis was performed using the JMP software (SAS Institute Inc.). All treatments were successful at generating colonies after 28 days of mOSKM transduction, with 32 colonies in bFGF (0.53% efficiency), 21 colonies in bFGF+2i (0.35% efficiency), 5 colonies in LIF (0.08% efficiency), and 3 colonies in LIF+2i (0.05% efficiency) treatments/groups. As an initial pluripotency test, all colonies were positive for AP activity at passage 3. The colonies were cultured for at least 25 passages (±200 days) except for those from the LIF+2i treatment, which were not able to remain viable after 15 passages. Gene expression analysis of the pluripotency (naïve and primed) biomarkers in biPSCs by RT-PCR revealed that colonies from the bFGF treatment were upregulated in NANOG, OCT4, (pluripotency biomarkers), and STELLA (naïve biomarker) (P<0.05) compared with bFGF+2i and LIF groups. There were no differences in expression of SOX2 (pluripotency biomarker gene) and naïve/primed biomarkers (OXT2, LIFr, and ESRRb) (P>0.05). Additionally, the relative abundance of mOSKM was not different between groups (P>0.05). For further pluripotency analysis, biPS colonies were tested for the invitro differentiation assay, and all colonies tested were able to form embryoid bodies. In conclusion, bovine fetal fibroblasts were successfully reprogrammed when using OSKM in all medium tested; however, LIF+2i treatment did not grow beyond 25 passages. Further tests should be performed to determine the pluripotency status of these biPSCs. We acknowledge FAPESP for funding (grant nos. 2012/50533-2, 2015/26816-5, and 2016/16841-2).

scholarly journals Reprogramming of Human Huntington Fibroblasts Using mRNA

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Antje Arnold ◽  
Yahaira M. Naaldijk ◽  
Claire Fabian ◽  
Henry Wirth ◽  
Hans Binder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Vectors ◽  
Viral Vector ◽  
Ips Cells ◽  
Expression Array ◽  
Healthy Donors ◽  
Pluripotency Gene ◽  
Cell Sources ◽  
Induced Pluripotent

The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.

scholarly journals Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

2014 ◽  
Vol 20 (23-24) ◽  
pp. 3154-3162 ◽  
Author(s):  
Minoru Tomizawa ◽  
Fuminobu Shinozaki ◽  
Yasufumi Motoyoshi ◽  
Takao Sugiyama ◽  
Shigenori Yamamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Episomal Vectors ◽  
Induced Pluripotent

scholarly journals Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

Stem Cells ◽  
2015 ◽  
Vol 33 (2) ◽  
pp. 392-402 ◽  
Author(s):  
Harald Lahm ◽  
Stefanie Doppler ◽  
Martina Dreßen ◽  
Astrid Werner ◽  
Klaudia Adamczyk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotency Gene ◽  
Induced Pluripotent

scholarly journals Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kim Hoa Ho ◽  
Annarita Patrizi
Keyword(s):  
Gene Expression ◽  
Choroid Plexus ◽  
Sensory Deprivation ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Experimental Conditions ◽  
Brain Repair ◽  
Expression Studies ◽  
Testing Algorithms ◽  
Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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