Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton’s jelly mesenchymal stem cells
Abstract:BackgroundWharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) are gaining increasing interest as an alternative source of stem cells for regenerative medicine applications. Definitive endoderm (DE) specification is a prerequisite for the development of vital organs such as liver and pancreas. Hence, efficient induction of the DE lineage from stem cells is crucial for subsequent generation of clinically relevant cell types. Here we present a defined 3D differentiation protocol of WJ-MSCs into DE cells.Meth… Show more
“…Summing up, formation of 3D cellular spheroids under FBS-free conditions was reported before, but mostly in applications other than therapeutic vascularization 39 , 47 , 55 . Here, we show that XF spheroids present no differences in terms of morphology, size, ECM deposition and OEC organization, as compared to FBS-grown spheroids, while exhibiting enhanced angiogenic potential.…”
Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.
“…Summing up, formation of 3D cellular spheroids under FBS-free conditions was reported before, but mostly in applications other than therapeutic vascularization 39 , 47 , 55 . Here, we show that XF spheroids present no differences in terms of morphology, size, ECM deposition and OEC organization, as compared to FBS-grown spheroids, while exhibiting enhanced angiogenic potential.…”
Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.
Stem cells constantly encounter as well as respond to a variety of signals in their microenvironment. Although the role of biochemical factors has always been emphasized, the significance of biophysical signals has not been studied until recently. Additionally, biophysical elements, like extracellular matrix (ECM) stiffness, can regulate functions of stem cells. In this study, we demonstrated that soft matrix with 1-10 kPa can induce neural differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). Importantly, we used a combination of soft matrix and bone morphogenetic protein receptor (BMPR) inhibition to promote neurogenic differentiation of hUC-MSCs. Furthermore, BMPR/SMADs occurs in crosstalk with the integrinβ1 downstream signaling pathway. In addition, BMPR inhibition plays a positive role in maintaining the undifferentiated state of hUC-MSCs on the hydrogel substrate. The results provide further evidence for the molecular mechanisms via which stem cells convert mechanical inputs into fateful decisions.
“…With a growing understanding of the advantages of 3D culture in the field of regenerative medicine, several protocols for the generation of 3D constructs under defined conditions have been developed, mostly for pluripotent stem cells (PSC) and MSC (Al Madhoun et al, ; Bazhanov, Mohammadipoor, & Bartosh, ; Duque‐Fernandez et al, ; Miranda, Fernandes, Diogo, & Cabral, ).…”
Section: Current Alternatives To the Use Of Fbs In Ec Culturementioning
confidence: 99%
“…lated from UCB and grown in 3D collagen matrices. In the presence of commercial platelet lysate, ECFC formed more vascular structures both in vitro and in vivo, as revealed by the quantification of perfused human microvessels, in comparison to ECFC grown in control scaffolds without platelet supplementation.With a growing understanding of the advantages of 3D culture in the field of regenerative medicine, several protocols for the generation of 3D constructs under defined conditions have been developed, mostly for pluripotent stem cells (PSC) and MSC (AlMadhoun et al, 2016;Bazhanov, Mohammadipoor, & Bartosh, 2017;Duque- Fernandez et al, 2016;Miranda, Fernandes, Diogo, & Cabral, 2016).Another humanized large-scale strategy for the propagation of ECFC was proposed byReinisch et al (2009). The cells were successfully isolated from both UCB and PB under XF conditions and further expanded to achieve clinically relevant doses of up to 150 million cells.The proposed XF formulation was based on the standard endothelial growth medium (EGM; basal medium containing GF cocktail), further supplemented with 10% human platelet lysate (HPL) pooled from a minimum of 40 donors, in place of the standard 5% FBS.…”
Two decades after the first report on endothelial progenitor cells (EPC), their key role in postnatal vasculogenesis and vascular repair is well established. The therapeutic potential of EPC and their growing use in clinical trials calls for the development of more robust, reproducible, and safer methods for the in vitro expansion and maintenance of these cells. Despite many limitations associated with its usage, fetal bovine serum (FBS) is still widely applied as a cell culture supplement. Although different approaches aiming at establishing FBS-free culture have been developed for many cell types, adequate solutions for endothelial cells, and for EPC in particular, are still scarce, possibly due to the multiple challenges that have to be faced when culturing these cells. In this review, we provide a brief overview on the therapeutic relevance of EPC and critically analyse the available literature on FBS-free endothelial cell culture methods, including xeno-free, serum-free, and chemically defined systems.
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