Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system

Abstract

Background: Pigs 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. Methods: iPS 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 pluripotencyrelated 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.

A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

“…Pig iPS cells with doxycyclineinducible piggyBac (PB) expression vectors encoding porcine 4 factors (OSKC) NANOG expression were absent [34]. Our previous work showed that pig PC-iPS with 4 factors (OSKC), NANOG is heterogeneously expressed in LCDMV culture medium [14]. Others report that in pig iPS cells generated by episomal vectors [32] and lentivirus vectors [47] separately containing 6 factors (OKSM+NANOG+LIN28) NANOG is expressed, but exogenous NANOG factor has not been silenced.…”

“…The pluripotency of NANOG tdTomato knock-in positive PC-iPS cells was corroborated in vitro with the following assays. NANOG knock-in positive PC-iPS cells were positive to AP staining (Additional file 3: Figure S2C), the method of AP staining is reference for our publication data [14]. Clustering analysis showed that knock-in positive PC-iPS cells could be clustered with pig EPS cells [12], but were separate from trophoblast cell (TE), inner cell mass (ICM), and early blastocyst (SB) [43] ( Fig.…”

“…Pig pericyte-derived induced pluripotent stem cells (PC-iPS cells) were cultured in a modified EPS culture system [14]. Cells were cultured in LCDMV, which consisted of a base medium containing 50% (v/v) DF12 (Gibco;10,565-018) and 50% (v/v) Neurobasal™ Medium (Gibco, 21103-049).…”

“…Recently, pig pluripotent stem cells (PSCs) were established from the inner cell mass of pig blastulas [11][12][13]. We found that induced pluripotent stem cells (iPSCs) from pigs express NANOG heterogeneously [14], as in mouse PSCs [15,16]. Various CRISPR/ Cas9 gene-editing strategies have been used to create reporter cell lines that accurately represent NANOG expression dynamics [16][17][18][19][20].…”

“…Pig pluripotent stem cells can be used in pig breeding, to model pig disease and to test pre-clinical regenerative medicines. Although pig expanded pluripotent stem cells [12], dome-shaped iPS cells [14], and ESCs [13] have recently been established, germline ESCs/iPSCs are not yet available. Many cytokines have been used to generate pig pluripotent stem cells, such as LIF [32][33][34], bFGF [35], and LIF and bFGF in combination [36,37].…”