3 years ago

Unrestricted Somatic Stem Cells, as a Novel Feeder Layer: Ex Vivo Culture of Human Limbal Stem Cells

Vahid Ezzatizadeh, Sayena Jabbehdari, Alireza Baradaran-Rafii, Ebrahim Shirzadeh, Saeed Heidari keshel
Ex vivo culture of limbal stem cells (LSCs) is a current promising approach for reconstruction of the ocular surface. In this context, 3T3 feeder layer cells (mouse embryo fibroblast) are generally utilized to maintain and expand LSCs. The aim of this study is to develop a novel culture method (animal-derived products free) to expand LSCs, using umbilical cord derived human unrestricted somatic stem cells (hUSSCs) instead of 3T3 cell with an emphasis on maintaining of the Stemness in LSCs. Using flow-cytometer, isolated hUSSCs were characterized for CD105, CD90, CD166, CD34, CD45, CD31 cell surface markers and their differentiation capability into adipogenic as well as osteogenic lineages were evaluated. In addition to colony-forming efficiency (CFE), epithelial lineage differentiation and karyotyping, LSC properties were evaluated for ABCG2, ΔNP63-α, CK19, CK3 and CK12 mRNA and protein expressions using quantitative RT-PCR (qRT-PCR) and immunocytochemistry, when these cells were co-cultured with hUSSCs (in comparison with 3T3 feeder layer). LSCs, co-cultured with hUSSCs, showed normal karyotype (46, XX), while they could efficiently form colony (86 ± 3) and display up-regulation of the genes associated with stemness and down-regulation of corneal epithelial differentiation genes. Consistent with 3T3 feeder cells, hUSSCs with spindle-shaped morphology and quick splitting up properties had ability to preserve the stem like-cell phenotype of LSCs. These findings were confirmed by qRT-PCR and flow-cytometer. Findings of present study suggest hUSSCs as a promising alternative method for 3T3 feeder layer cells, to preserve growth and stemness of LSCs ex vivo culture.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/jcb.26434

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.