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Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients.

ABSTRACT: Sickle cell disease (SCD) is a monogenic red cell disorder associated with multiple vascular complications, microvessel injury and wound-healing deficiency. Although stem cell transplantation with bone marrow-derived mesenchymal stem cells (BMSC) can promote wound healing and tissue repair in SCD patients, therapeutic efficacy is largely dependent on the paracrine activity of the implanted BM stromal cells. Since in vitro expansion and culture conditions are known to modulate the innate characteristics of BMSCs, the present study investigated the effects of normoxic and hypoxic cell-culture preconditioning on the BMSC secretome, in addition to the expression of paracrine molecules that induce angiogenesis and skin regeneration. BMSCs derived from SCD patients were submitted to culturing under normoxic (norCM) and hypoxic (hypoCM) conditions. We found that hypoxically conditioned cells presented increased expression and secretion of several well-characterized trophic growth factors (VEGF, IL8, MCP-1, ANG) directly linked to angiogenesis and tissue repair. The hypoCM secretome presented stronger angiogenic potential than norCM, both in vitro and in vivo, as evidenced by HUVEC proliferation, survival, migration, sprouting formation and in vivo angiogenesis. After local application in a murine wound-healing model, HypoCM showed significantly improved wound closure, as well as enhanced neovascularization in comparison to untreated controls. In sum, the secretome of hypoxia-preconditioned BMSC has increased expression of trophic factors involved in angiogenesis and skin regeneration. Considering that these preconditioned media are easily obtainable, this strategy represents an alternative to stem cell transplantation and could form the basis of novel therapies for vascular regeneration and wound healing in individuals with sickle cell disease.

SUBMITTER: Ribeiro TO

PROVIDER: S-EPMC6821040 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients.

Ribeiro Tiago O TO Silveira Brysa M BM Meira Mercia C MC Carreira Ana C O ACO Sogayar Mari Cleide MC Meyer Roberto R Fortuna Vitor V

PloS one 20191030 10

Sickle cell disease (SCD) is a monogenic red cell disorder associated with multiple vascular complications, microvessel injury and wound-healing deficiency. Although stem cell transplantation with bone marrow-derived mesenchymal stem cells (BMSC) can promote wound healing and tissue repair in SCD patients, therapeutic efficacy is largely dependent on the paracrine activity of the implanted BM stromal cells. Since in vitro expansion and culture conditions are known to modulate the innate characte ...[more]

PMID: 31665139

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Project description:OBJECTIVE:Regenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. METHODS:The secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. RESULTS:Both BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. CONCLUSIONS:PL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

Dataset Information

Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients.

Publications

Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients.

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