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Background <p>Despite the relevance of induced pluripotent stem cells (iPSCs) technology for biotechnological applications in cattle, the generation of bona fide bovine iPSCs (biPSCs) remains challenging. This study aimed to evaluate the use of a non-integrating Sendai virus system carrying polycistronic human <i>KLF4</i>–<i>OCT3/4</i>–<i>SOX2</i>, <i>c-MYC</i>, and <i>KLF4</i> to reprogram bovine fetal mesenchymal stem/stromal cells (MSCs) derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) into biPSCs.</p> Methods and results <p>AT-MSCs, BM-MSCs, and control fibroblasts (FBs) were isolated from bovine fetuses (<i>n</i> = 9; 8 months of gestation). A CytoTune™-iPS 2.0 Sendai kit was used for reprogramming AT-MSCs, BM-MSCs and FBs. Cell culture samples were collected on days 0, 7, and 28 post-transduction for pluripotency and epigenetic gene expression analysis by quantitative PCR (qPCR) and on day 28 for assessment of pluripotency markers by immunofluorescence and alkaline phosphatase (AP) activity. Cells formed colonies with undefined margins and low AP activity. Endogenous bovine OCT4, SOX2, and NANOG proteins were detected in the nuclei and cytoplasm of FBs, AT-MSCs, and BM-MSCs. However, <i>OCT4</i> gene expression was detected only in FBs. Gene expression of DNA methyltransferase <i>DNMT3B</i> and histone demethylase <i>KDM6</i> were increased (<i>P</i> &lt; 0.05) in AT-MSCs and FBs, respectively.</p> Conclusions <p>Insufficient activation of core pluripotency (i.e., endogenous OCT4) and undefined colony morphology indicate that reprogramming was not achieved in AT-MSCs, BM-MSCs, or FBs following Sendai virus transduction. Upregulation of <i>DNMT3A</i> in AT-MSCs and <i>EZH2</i> in FBs suggests reduced demethylation of DNA and histones, which may account for the failed reprogramming of these cells.</p>

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Sendai virus-based human transcriptional gene delivery system fails to reprogram bovine mesenchymal stromal cells (MSC) into induced pluripotent stem cells (iPSC)

  • 期刊论文

  • 发表日期：

    2026年5月22日

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  • Blanca Quiroga,
  • Barbara Leiva,
  • Carlos Díaz,
  • Francisco J. Morera,
  • Luis Paiva,
  • Cristian G. Torres,
  • Víctor H. Parraguez,
  • Oscar A. Peralta

摘要

Background

Despite the relevance of induced pluripotent stem cells (iPSCs) technology for biotechnological applications in cattle, the generation of bona fide bovine iPSCs (biPSCs) remains challenging. This study aimed to evaluate the use of a non-integrating Sendai virus system carrying polycistronic human KLF4–OCT3/4–SOX2, c-MYC, and KLF4 to reprogram bovine fetal mesenchymal stem/stromal cells (MSCs) derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) into biPSCs.

Methods and results

AT-MSCs, BM-MSCs, and control fibroblasts (FBs) were isolated from bovine fetuses (n = 9; 8 months of gestation). A CytoTune™-iPS 2.0 Sendai kit was used for reprogramming AT-MSCs, BM-MSCs and FBs. Cell culture samples were collected on days 0, 7, and 28 post-transduction for pluripotency and epigenetic gene expression analysis by quantitative PCR (qPCR) and on day 28 for assessment of pluripotency markers by immunofluorescence and alkaline phosphatase (AP) activity. Cells formed colonies with undefined margins and low AP activity. Endogenous bovine OCT4, SOX2, and NANOG proteins were detected in the nuclei and cytoplasm of FBs, AT-MSCs, and BM-MSCs. However, OCT4 gene expression was detected only in FBs. Gene expression of DNA methyltransferase DNMT3B and histone demethylase KDM6 were increased (P < 0.05) in AT-MSCs and FBs, respectively.

Conclusions

Insufficient activation of core pluripotency (i.e., endogenous OCT4) and undefined colony morphology indicate that reprogramming was not achieved in AT-MSCs, BM-MSCs, or FBs following Sendai virus transduction. Upregulation of DNMT3A in AT-MSCs and EZH2 in FBs suggests reduced demethylation of DNA and histones, which may account for the failed reprogramming of these cells.