RESUMEN
Human adipose tissue-derived stem cells (hASC) secretome display various therapeutically relevant effects in regenerative medicine, such as induction of angiogenesis and tissue repair. The benefits of hASC secretome are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in host cells. However, the composition and the innate characteristics of hASC secretome can be highly variable depending on the culture conditions. Here, we evaluated the combined effect of serum-free media and hypoxia preconditioning on the hASCs secretome composition and biological effects on angiogenesis and wound healing. The hASCs were cultured in serum-free media under normoxic (NCM) or hypoxic (HCM) preconditioning. The proteomic profile showed that pro- and anti-antiangiogenic factors were detected in NCM and HCM secretomes. In vitro studies demonstrated that hASCs secretomes enhanced endothelial proliferation, survival, migration, in vitro tube formation, and in vivo Matrigel plug angiogenesis. In a full-thickness skin-wound mouse model, injection of either NCM or HCM significantly accelerated the wound healing. Finally, hASC secretomes were potent in increasing endothelial density and vascular coverage of resident pericytes expressing NG2 and nestin to the lesion site, potentially contributing to blood vessel maturation. Overall, our data suggest that serum-free media or hypoxic preconditioning enhances the vascular regenerative effects of hASC secretome in a preclinical wound healing model.
Asunto(s)
Células Madre Mesenquimatosas , Secretoma , Ratones , Animales , Humanos , Pericitos , Medio de Cultivo Libre de Suero , Proteómica , Tejido Adiposo/metabolismoRESUMEN
The potential use of bone marrow mesenchymal stromal cells (BM-MSCs) for the treatment of osteonecrosis in sickle cell disease (SCD) patients is increasing. However, convenient BM-MSC quantification and functional property assays are critical factors for cell-based therapies yet to be optimized. This study was designed to quantify the MSC population in bone marrow (BM) samples from SCD patients with osteonecrosis (SCD group) and patients with osteoarticular complications not related to SCD (NS group), using flow cytometry for CD271+CD45-/low cell phenotype and CFU-F assay. We also compared expanded BM-MSC osteogenic differentiation, migration, and cytokine secretion potential between these groups. The mean total cell number, CFU-F count, and CD271+CD45-/low cells in BM mononuclear concentrate were significantly higher in SCD than in NS patients. A significant correlation between CD271+CD45-/low cell number and CFU-F counts was found in SCD (r = 0.7483; p = 0.0070) and NS (r = 0.7167; p = 0.0370) BM concentrates. An age-related quantitative reduction of CFU-F counts and CD271+CD45-/low cell number was noted. Furthermore, no significant differences in the morphology, replicative capacity, expression of surface markers, multidifferentiation potential, and secretion of cytokines were found in expanded BM-MSCs from SCD and NS groups after in vitro culturing. Collectively, this work provides important data for the suitable measurement and expansion of BM-MSC in support to advanced cell-based therapies for SCD patients with osteonecrosis.
RESUMEN
Recent evidence suggests that abnormalities involving CD4+T lymphocytes are associated with the pathophysiology of osteonecrosis (ON); however, few studies have addressed the CD4+T cells in ON related to sickle cell disease (SCD/ON). In addition, T cells producing multiple cytokines simultaneously are often present in the inflammatory milieu and may be implicated in the immune response observed in SCD/ON. In the present study, we aimed to characterize the functional status of CD4+T cells in SCD by simultaneously determining the frequency of IFN-γ +, IL-4+, and IL-17+ CD4+T in cell cultures under exogenous stimuli. Peripheral blood mononuclear cells (PB-MNCs) from 9 steady-state SCD patients, 15 SCD/ON patients, and 19 healthy controls had functional status of CD4+T cells analyzed. Bone marrow mononuclear cells (BM-MNCs) from 24 SCD/ON patients (SCD BM) and 18 patients with ON not related to SCD (non-SCD BM) were also analyzed. We found that PB-MNC of SCD patients with or without ON presented significantly reduced TCD4+, TCD8+, and TCD4+ naïve cell frequencies and increased frequency of circulating CD4+T cells able to simultaneously produce IFN-γ +/IL4+ and IL-17+/IL4+ compared to healthy controls. Conversely, the polyclonal stimulation of BM-MNC induced an increased frequency of CD4+IFN-γ + and CD4+IL-17+ in SCD BM compared to non-SCD BM. The increased proportion of CD4+ T cells able to produce a broad spectrum of proinflammatory cytokines after a strong stimulus indicates that the immune system in SCD/ON patients presents an expressive pool of partially differentiated cells ready to take on effector function. It is possible that this increased subpopulation may extend to inflammatory sites of target organs and may contribute to the maintenance of inflammation and the pathophysiology of osteonecrosis in sickle cell disease.
Asunto(s)
Anemia de Células Falciformes/inmunología , Anemia de Células Falciformes/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Osteonecrosis/inmunología , Osteonecrosis/metabolismo , Adolescente , Adulto , Femenino , Células Madre Hematopoyéticas/metabolismo , Hemoglobinas/metabolismo , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
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.