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An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

ABSTRACT: Cell-free therapy based on conditioned medium derived from mesenchymal stromal cells (MSCs) has gained attention in the field of protective and regenerative medicine. However, the exact composition and properties of MSC-derived conditioned media can vary greatly depending on multiple parameters, which hamper standardization. In this study, we have optimized a procedure for preparation of conditioned medium starting from efficient isolation, propagation and characterization of MSCs from human umbilical cord, using a culture medium supplemented with human platelet lysate as an alternative source to fetal bovine serum. Our procedure successfully maximizes the yield of viable MSCs that maintain canonical key features. Importantly, under these conditions, the compositional profile and biological effects elicited by the conditioned medium preparations derived from these MSC populations do not depend on donor individuality. Moreover, approximately 120 L of conditioned medium could be obtained from a single umbilical cord, which provides a suitable framework to produce industrial amounts of toxic-free conditioned medium with predictable composition.

SUBMITTER: Acuto S

PROVIDER: S-EPMC10580810 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

Acuto Santina S Lo Iacono Melania M Baiamonte Elena E Lo Re Rosa R Maggio Aurelio A Cavalieri Vincenzo V

Frontiers in molecular biosciences 20231002

Cell-free therapy based on conditioned medium derived from mesenchymal stromal cells (MSCs) has gained attention in the field of protective and regenerative medicine. However, the exact composition and properties of MSC-derived conditioned media can vary greatly depending on multiple parameters, which hamper standardization. In this study, we have optimized a procedure for preparation of conditioned medium starting from efficient isolation, propagation and characterization of MSCs from human umb ...[more]

PMID: 37854039

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Project description:BACKGROUND:Keloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known. It has been hypothesized that keloids behave like benign tumors because of their uncontrolled growth. The present study evaluated the tumoricidal properties of human Wharton's jelly stem cell-conditioned medium (hWJSC-CM) on fresh Asian keloid cells (AKCs). METHODS:Human Wharton's jelly stem cells (hWJSCs) and AKCs were isolated based on our previous methods. hWJSCs and human skin fibroblasts (HSF) (controls) were used to collect hWJSC-CM and HSF-conditioned medium (HSF-CM). AKCs were treated with hWJSC-CM and HSF-CM in vitro and in vivo in a human keloid xenograft SCID mouse model. The inhibitory effect of hWJSC-CM on AKCs was tested in vitro using various assays and in vivo for attenuation/abrogation of AKC tumors created in a xenograft mouse model. RESULTS:qRT-PCR analysis showed that the genes FN1, MMP1, and VCAN were significantly upregulated in AKCs and ANXA1, ASPN, IGFBP7, LGALS1, and PTN downregulated. AKCs exposed to hWJSC-CM in vitro showed significant decreases in cell viability and proliferation, increases in Annexin V-FITC+ cell numbers, interruptions of the cell cycle at Sub-G1 and G2/M phases, altered CD marker expression, downregulated anti-apoptotic-related genes, and upregulated pro-apoptotic and autophagy-related genes compared to controls. When AKCs were administered together with hWJSC-CM into immunodeficient mice there were no keloid tumors formed in 7 mice (n =?10) compared to the untreated control mice. When hWJSC-CM was injected directly into keloid tumors created in mice there were significant reductions in keloid tumor volumes and weights in 30?days. CONCLUSIONS:hWJSC-CM inhibited the growth of AKCs in vitro and in xenograft mice, and it may be a potential novel treatment for keloids in the human. The specific molecule(s) in hWJSC-CM that induce the anti-keloid effect need to be identified, characterized, and tested separately in larger preclinical and clinical studies.

Project description:BackgroundWharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17β-estradiol and 8-Br-cAMP on the differentiation system.MethodsWJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17β-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17β-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells.Results17β-estradiol at 1 μM downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected.Conclusions17β-estradiol at 1 μM is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

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An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

Publications

An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

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