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Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.

ABSTRACT: We describe highly effective adhesion culture of human pluripotent stem cells (hPSCs) using laminin fragments without precoating. Culture substrates have been generally thought to exert a cell adhesion effect when they are precoated onto culture vessels. However, simple addition of laminin fragments to a cell suspension during passaging accelerated the adhesion of single dissociated hPSCs onto culture vessels that were not precoated with any culture substrate. Interestingly, similar to conventional precoating, the uncoated addition of laminin fragments supported robust adhesion of single hPSCs and maximum adhesion at a much lower concentration compared with precoating. Similar to precoating laminin fragments, hPSCs seeded with uncoated laminin fragments grew well without cell detachment and maintained pluripotency after continuous subculture. We tested other culture substrates, including full-length laminin and vitronectin, to support hPSC adhesion in the uncoated manner, but only laminin fragments had the potential for application in the uncoated manner. This cost-effective and time-efficient method may contribute to expansion of culture of hPSCs and accelerate the development of regenerative medicine using hPSCs.

SUBMITTER: Miyazaki T

PROVIDER: S-EPMC5278368 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.

Miyazaki Takamichi T Isobe Takehisa T Nakatsuji Norio N Suemori Hirofumi H

Scientific reports 20170130

We describe highly effective adhesion culture of human pluripotent stem cells (hPSCs) using laminin fragments without precoating. Culture substrates have been generally thought to exert a cell adhesion effect when they are precoated onto culture vessels. However, simple addition of laminin fragments to a cell suspension during passaging accelerated the adhesion of single dissociated hPSCs onto culture vessels that were not precoated with any culture substrate. Interestingly, similar to conventio ...[more]

PMID: 28134277

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Project description:BackgroundPigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.MethodsiPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.ResultsIn this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.ConclusionsOur findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Dataset Information

Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.

Publications

Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.

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