Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition.

Arterial diseases continue to pose a major health concern but in vitro studies are limited because explanted cells can exhibit poor proliferative capacity and a loss of specificity. Here, we find that two transcription factors, MYCN and SOX17, induce and indefinitely expand in culture precursors of human arterial endothelial cells (expandable arterial endothelial precursors [eAEPs]). The eAEPs are derived from CD34+ cells found in umbilical cord blood or adult bone marrow. Independent eAEP lines differ in their proclivity to undergo an endothelial-to-mesenchymal transition (EndoMT), a hallmark event in a broad array of vascular diseases and disorders. Some cell lines spontaneously become mesenchymal over time in culture, an effect exacerbated by inhibition of the fibroblast growth factor receptor, while others do not readily convert. These distinctions were exploited to identify genes that correlate with resistance to an EndoMT and to elucidate transcriptional changes that underpin the transition.

Pax3-induced expansion enables the genetic correction of dystrophic satellite cells.

BACKGROUND: Satellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as self-renewal and long-term regenerative potential. METHODS: We develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice. RESULTS: Here, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human µDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury. CONCLUSION: These results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties.