Systematic in vitro and in vivo characterization of Leukemia‐inhibiting factor‐ and Fibroblast growth factor‐derived porcine induced pluripotent stem cells

Secher, Jan Bojsen-Møller; Ceylan, Ahmet; Mazzoni, Gianluca; Mashayekhi, Kaveh; Li, Tong; Muenthaisong, Suchitra; Nielsen, Troels Tolstrup; Liu, Dong; Li, Shengting; Petkov, Stoyan; Cirera, Susanna; Luo, Yonglun; Thombs, Lori A.; Kadarmideen, Haja N.; Dinnyés, András; Bolund, Lars; Schmidt, M.; Callesen, Henrik; Hyttel, Poul; Freude, Kristine

doi:10.1002/mrd.22771

Cited by 16 publications

(14 citation statements)

References 62 publications

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“…Immunocytochemistry revealed the nuclear presence of the transcription factor Nanog in fibroblast spheroids only; this not only corroborates translation of normally silenced gene in differentiated fibroblasts, [20] but also allows a first glimpse into the percentage of cells in such a spheroid that might spontaneous respond to the mechanical stimulus that a spheroid poses. [22] CD7 (84-fold) has been observed in certain stages of pluripotent human stem cells. Considering the natural heterogeneity of primary fibroblasts directly isolated from tissue, this percentage is surprisingly high, given current forced transduction efficiencies in monolayer cultures to induce pluripotency (0.1-3% efficiency [21] ).…”

Section: Discussionmentioning

confidence: 99%

Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes

Raghunath²,

Lee

2019

Adv. Biosys.

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approaches have been adopted to coax cells in single cell suspension to aggregate and form spheroids, such as i) hanging drop culture, ii) growing cells on low-attachment culture surfaces, and iii) culturing cells in rotating bioreactors. The principle of these methods is to prevent cells from adhering to the cell culture vessel surface and instead encourage the cells to adhere to each other. This would then force the cells to form aggregates/spheroids, establish close cell-cell contacts and communication. Spheroid cultures are considered to be better suited for studying a wide range of cellular activities, such as tissue development and disease. [3] In the past decade, the use of spheroid culture to investigate cancer biology in vitro has increased significantly because it morphologically resembles a tumor. It has been reported that cancer cell spheroids mimic a more "realistic" cellular response to radioactive [4] and chemical [5] treatments than cancer cells maintained as 2-dimensional (2D) monolayer culture. Thus, cancer spheroids are emerging as a preferable model for identifying and translating new types of cancer therapies.Several studies have highlighted that the expression of numerous genes change when the same cell type is grown as spheroid instead of monolayer culture. These genes relate to cell differentiation and cell reprogramming, particularly key pluripotency genes Oct4, Sox2, and Nanog. This was observed in mouse embryonic stem cells, [6] human mesenchymal stem cells [7] (MSCs), human cancer cell lines, [8] and mouse fibroblasts. [9] Research has also revealed that spheroid cultures favors mesenchymal-to-epithelial transition (MET), maintenance and even induction of epithelial phenotypes in various cell types. [9][10][11] MET is an essential initiation step in stem cell reprogramming and thus dedifferentiation of mesenchymal lineage cell types. [12] Indeed, MSC spheroids have been reported to acquire enhanced multipotency so that the MSCs could differentiate into more diverse cell types. Cesarz and Tamama claimed that MSC cultured as spheroids were capable to differentiating into more lineages than otherwise possible when cultured as monolayer. [13] Together, these findings suggest the use spheroid cultures might induce spontaneous induction of dedifferentiation, and initial expression of pluripotency markers, and this might be useful at a later stage to increase reprogramming efficiency in the iPSC field.Suspension spheroid cultures of anchorage-dependent cell types have been widely used in cancer and stem cell research, as well as for producing organoids. It is believed that the 3-dimensional spheroid presents cells with a more physiological microenvironment to grow so that they behave more like cells in vivo, which is lacking in conventional 2-dimensional monolayer cultures. Recently, it has been reported that cancer cells grown as spheroids could express stem cell-associated genes. Hence, it is investigated whether normal mouse and human fibroblasts cultured as spheroids could also be induced...

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Section: Discussionmentioning

confidence: 99%

Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes

Raghunath²,

Lee

2019

Adv. Biosys.

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“…These piPSCs resemble hESCs in the expression of SSEA-4 and SSEA-1 (23), presumably due to the presence of LIF. piPSCs grown in the presence of LIF express SSEA-4 while piPSCs grown in the presence of FGF express SSEA-1 (24; 25; 26). This was further supported by the low expression of SSEA-4 observed in the LIF-independent piPSC line used in the current study.…”

Section: Discussionmentioning

confidence: 99%

“…NESTIN represents a reliable marker for neural progenitor cell differentiation from porcine cells (30; 31; 32). ACTA2 , the gene encoding for the protein αSMA, was selected as a reliable marker for mesodermal differentiation for piPSCs (26), as well as hESC differentiation towards smooth muscle cells (33). GATA6 has been shown in previous studies to be expressed upon endodermal lineage differentiation of piPSCs (30; 31) and is considered to play an integral role in the formation of human definitive endoderm from pluripotent stem cells (34).…”

Section: Discussionmentioning

confidence: 99%

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

Burrell

Dardari

Goldsmith

et al. 2019

Preprint

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45Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and 46 the development of therapies, as they can proliferate indefinitely under defined conditions and 47 differentiate into any cell type in the body. Large scale expansion of cells is limited in adherent 48 culture, making it difficult to obtain adequate cell numbers for research. It has been previously 49 shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem 50 cells. Pigs are important pre-clinical models for stem cell research. Therefore, this study 51 investigated the use of SSBs as an alternative culture method for the expansion of iPSCs. Using 52 an established porcine iPSC line as well as a new cell line derived and characterized in the current 53 study, we report that porcine iPSCs (piPSCs) can grow in SSB while maintaining characteristics 54 of pluripotency and karyotypic stability similar to cells grown in traditional two-dimensional static 55 culture. This culture method provides a suitable platform for scale up of cell culture to provide 56 adequate cell numbers for future research applications involving porcine induced pluripotent stem 57 cells. 58 59 60 61 62 63 64 65 66 68 Pluripotent stem cells (PSCs) are defined by two main features: the ability to proliferate 69 indefinitely in an undifferentiated state under defined conditions, and the potential to become any 70 cell type arising from the three germ layers, endoderm, ectoderm and mesoderm, in the body. 71 Human and mouse embryonic stem cell (ESC) lines have significantly contributed to the progress 72 in biomedical research and cell therapy development (1; 2), however, the ethical and safety 73 concerns surrounding the use of human embryonic stem cells (hESCs) have limited their potential 74 applications (3). The emergence of induced pluripotent stem cells (iPSCs) as an alternative source 75 of cells that recapitulate the phenotype and function of ESCs, helped overcome these concerns and 76 energized the stem cell research field (4; 5). 77Outbred, large animal models that are closer in size, longevity and physiology to humans are 78 essential to expand our knowledge gained from rodents and facilitate translation of stem cell-based 79 approaches to human applications. iPSCs have been derived from large ungulate species including 80 pigs that share many physiological characteristics with humans, making the pig is a powerful 81 model for biomedical research and drug testing (6). 82Large numbers of cells are required for many research and therapeutic applications, which can be 83 challenging to obtain through conventional two-dimensional static culture systems in tissue culture 84 plates and flasks. Two-dimensional culture systems have limited surface area, often require a large 85 number of culture vessels and can be labor intensive. This additional handling can lead to cell loss 86 and increased risk for contamination. Moreover, the use of numerous plates and flasks may 87 introduce culture heterogeneity ...

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“…After filtration of low-quality reads, as shown in the box plot distributions of Figure S1, the quality scores across all bases were in the high confidence range. The filtered sequence reads and previously reported data for pig preimplantation embryos [9] were aligned to the pig reference genome (Sscrofa10.2) with 80.5-92.2% mapping rates (Table S2). After the mapping, gene expression levels were calculated by the HTSeq-count program (Fig.…”

Section: Quality Control Assessmentmentioning

confidence: 99%

Transcriptome profiling of pluripotent pig embryonic stem cells originating from uni- and biparental embryos

Choi

Lee

et al. 2020

BMC Res Notes

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Objectives: Pig pluripotent stem cells have tremendous potential because the pig is a valuable animal as both an agricultural resource and as a preclinical model of human therapy. To date, a lack of understanding of pig pluripotency has inhibited the derivation of embryonic stem cells (ESCs) and transgene-free induced pluripotent stem cells. Therefore, there has been no accessible or reliable transcriptome data for researching the genuine pig pluripotency network. Our previous study isolated authentic pig ESCs, which had teratoma-forming and direct differentiation ability, that were derived by activating the FGF2, ACTIVIN A, and WNT pathways. Here, we aimed to provide detailed information on transcriptome data of the newly derived pig ESCs and perform a comparative analysis with pig preimplantation embryo transcriptomes in a public database. Data description: The transcriptome data of ESCs derived from in vitro fertilized and parthenogenetic embryos were generated by HiSeq 2500. Then, differentially expressed genes (DEGs) from each sample were compared with fibroblasts, and gene expression profiling was carried out for comparative analysis. Our data, as the first transcriptome dataset for genuine pig pluripotent cells, could be a general reference for explaining the molecular mechanism of species-specific pluripotency and improving understanding of the embryo development of domestic animals.

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Systematic in vitro and in vivo characterization of Leukemia‐inhibiting factor‐ and Fibroblast growth factor‐derived porcine induced pluripotent stem cells

Cited by 16 publications

References 62 publications

Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes

Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

Transcriptome profiling of pluripotent pig embryonic stem cells originating from uni- and biparental embryos

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