A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement

Abstract

IntroductionMesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes.MethodsIn this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement.ResultsWe showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum.ConclusionsThis protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt540) contains supplementary material, which is available to authorized users.

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review

“…VBD concentrations added to culture medium ranged from 0.5 to 30% (54)(55)(56). Overall, VBD allowed for either, maintenance of MCS's fibroblast-like morphology (17,43,57), high proliferation (58-60), high colony-formation ability (52,61,62) and maintenance of multipotency (63)(64)(65)(66)(67). A linear dose-dependent response regarding medium concentration was not observed for evaluated VBD (54,56,68).…”

“…HPL presented a high growth factors and cytokines content (74)(75)(76)(77). HPL seemed to be better than FBS for ex vivo MSC expansion (52,72,78,79) since HPL-expanded MSC did not present telomerase shortening (68).…”

“…Most of the included studies described protocols relying on the platelets lyse after freeze-thaw cycles to obtain VBDs. Bone marrow mesenchymal stem cells (BMSC) were presented in most of this studies (Table 5); adipose stem cells -ASC (24,(41)(42)(43)(44)(45)(46), dental pulp stem cells -DPSC (47,48), umbilical cord stem cells -UCST (22,25,(49)(50)(51)(52) and orbital fat-derived stem cells (53) were also tested. VBD concentrations added to culture medium ranged from 0.5 to 30% (54)(55)(56).…”

“…Besides, MSC expanded in HPL seems presented immunomodulatory ability (52,55,71,72) despite a decrease of ability of inhibition NK and T-cells has also been observed (73). Platelets lyse was triggered by either, freeze-thaw cycles (1 to 5), ultrasound (63) or chemical treatment (43).…”

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

“…Both cell lines were grown in DMEM supplemented with 10% FBS and antibiotics up to a 15 passage. All isolations of primary cells were approved by FLENI Ethic Committee after reviewing the research protocol (Luzzani et al, 2015). …”