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VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion.

ABSTRACT: The very small embryonic-like stem cells (VSELs) are known as a subset of adult pluripotent stem cells able to differentiate to all three germ layers. However, their small number and quiescence restrict the possibility of their use in cell therapy. In the present study, we first delineate different subpopulation of VSELs from human cord blood CD34+ cells to define their purity. We next determine genes expression levels in the whole transcriptome of VSELs expressing the pluripotent marker NANOG and control cells under the steady state condition. We found that more than a thousand of genes are downregulated in VSELs, as well as many membrane receptors, cells signaling molecules and CDKs mRNAs. In addition, we observed discordance in some pluripotent genes expression levels with embryonic stem cells (ESCs), which could explain VSELs quiescence. We then evaluate VSELs capacity to expand and differentiate in vitro in specific and appropriate media. After 12 days culture in specific medium containing a pyrimidoindole derivative (UM171), VSELs were significantly expanded for the first time without feeder cells and importantly preserve their capacities to differentiate into hematopoietic and endothelial cells. Interestingly, this stimulation of VSELs self-renewal restores the expression of some downregulated genes known as key regulators of cell proliferation and differentiation. The properties of such pluripotent expanded cells make them a potential candidate in regenerative medicine.

SUBMITTER: Lahlil R

PROVIDER: S-EPMC6013546 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion.

Lahlil Rachid R Scrofani Maurice M Barbet Romain R Tancredi Céline C Aries Anne A Hénon Philippe P

Stem cell reviews and reports 20180801 4

The very small embryonic-like stem cells (VSELs) are known as a subset of adult pluripotent stem cells able to differentiate to all three germ layers. However, their small number and quiescence restrict the possibility of their use in cell therapy. In the present study, we first delineate different subpopulation of VSELs from human cord blood CD34+ cells to define their purity. We next determine genes expression levels in the whole transcriptome of VSELs expressing the pluripotent marker NANOG a ...[more]

PMID: 29736843

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Project description:BackgroundSkin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium.MethodsConditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium.ResultsConditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers.ConclusionsOur results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.

Dataset Information

VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion.

Publications

VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion.

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