Steps towards standardized in vitro assessment of immunomodulatory equine mesenchymal stromal cells prior to clinical application

The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1β is one of the most abundant inflammatory mediators in this process and crucially affects the expression of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this study we aimed to investigate, for the first time, how IL-1β induced expression of MMPs, TIMPs and how IL-1β in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1β in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, MMP-2, TIMP-1 and IL-1β expression levels were measured. STS alone had no influence on the basal expression of investigated target genes, whereas IL-1β caused increased expression of these genes. In combination, they increased the gene and protein expression of MMP-1 and the gene expression of MMP-2 after 24 h. After 6 h, STS reduced IL-1β-induced MMP-1 synthesis and MMP-2 gene expression. IL-1β-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both time points, the IL-1β-induced gene expression of IL-1β was increased. Additionally, this study showed that fetal bovine serum (FBS) caused an overall suppression of IL-1β-induced expression of MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1β-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a more inflammatory and destructive response of hPDL-MSCs when using a static application form and that this response is highly influenced by the presence of FBS in vitro.

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Growth characteristics of human bone marrow mesenchymal stromal cells at cultivation on synthetic polyelectrolyte nanofilms in vitro

Heliyon ◽

10.1016/j.heliyon.2021.e06517 ◽

2021 ◽

Vol 7 (3) ◽

pp. e06517

Author(s):

Lyudmila M. Mezhevikina ◽

Dmitriy A. Reshetnikov ◽

Maria G. Fomkina ◽

Nurbol O. Appazov ◽

Saltanat Zh. Ibadullayeva ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Growth Characteristics ◽

Human Bone ◽

Human Bone Marrow

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3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02315-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Diego Noé Rodríguez-Sánchez ◽

Giovana Boff Araujo Pinto ◽

Luciana Politti Cartarozzi ◽

Alexandre Leite Rodrigues de Oliveira ◽

Ana Livia Carvalho Bovolato ◽

...

Keyword(s):

Growth Factor ◽

Sciatic Nerve ◽

Nerve Injury ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Multipotent Mesenchymal Stromal Cells ◽

Sciatic Nerve Injury ◽

Motor Deficits ◽

3D Printed

Abstract Background Nerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration. Methods 3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve. Results The inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair. Conclusions 3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

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Extracellular Vesicles Are More Potent Than Adipose Mesenchymal Stromal Cells to Exert an Anti-Fibrotic Effect in an In Vitro Model of Systemic Sclerosis

International Journal of Molecular Sciences ◽

10.3390/ijms22136837 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6837

Author(s):

Pauline Rozier ◽

Marie Maumus ◽

Claire Bony ◽

Alexandre Thibault Jacques Maria ◽

Florence Sabatier ◽

...

Keyword(s):

Systemic Sclerosis ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

In Vitro Model ◽

Specific Treatment ◽

Complex Disorder ◽

Molecular Features ◽

Vitro Model

Systemic sclerosis (SSc) is a complex disorder resulting from dysregulated interactions between the three main pathophysiological axes: fibrosis, immune dysfunction, and vasculopathy, with no specific treatment available to date. Adipose tissue-derived mesenchymal stromal cells (ASCs) and their extracellular vesicles (EVs) have proved efficacy in pre-clinical murine models of SSc. However, their precise action mechanism is still not fully understood. Because of the lack of availability of fibroblasts isolated from SSc patients (SSc-Fb), our aim was to determine whether a TGFβ1-induced model of human myofibroblasts (Tβ-Fb) could reproduce the characteristics of SSc-Fb and be used to evaluate the anti-fibrotic function of ASCs and their EVs. We found out that Tβ-Fb displayed the main morphological and molecular features of SSc-Fb, including the enlarged hypertrophic morphology and expression of several markers associated with the myofibroblastic phenotype. Using this model, we showed that ASCs were able to regulate the expression of most myofibroblastic markers on Tβ-Fb and SSc-Fb, but only when pre-stimulated with TGFβ1. Of interest, ASC-derived EVs were more effective than parental cells for improving the myofibroblastic phenotype. In conclusion, we provided evidence that Tβ-Fb are a relevant model to mimic the main characteristics of SSc fibroblasts and investigate the mechanism of action of ASCs. We further reported that ASC-EVs are more effective than parental cells suggesting that the TGFβ1-induced pro-fibrotic environment may alter the function of ASCs.

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871 In vitro immunoregulatory effect from cervical cancer derived mesenchymal stromal cells over molecules expression in monocyte derived macrophage polarization

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0871 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A923-A923

Author(s):

Víctor Cortés-Morales ◽

Juan Montesinos ◽

Luis Chávez-Sánchez ◽

Sandra Espíndola-Garibay ◽

Alberto Monroy-García ◽

...

Keyword(s):

Cervical Cancer ◽

Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Macrophage Polarization ◽

Induction Medium ◽

M2 Macrophage ◽

List Type ◽

M2 Phenotype

BackgroundMacrophages are immunological cells that sense microenvironmental signals that may result in the polarized expression of either proinflammatory (M1) or anti-inflammatory (M2) phenotype.1 Macrophages M2 are present in tumoral microenvironment and their presence in patients with cervical cancer (CeCa) is related with less survival.2Mesenchymal Stromal Cells (MSCs) are also present in tumor microenvironment of cervical cancer (CeCa-MSC), which have shown immunoregulatory effects over CD8 T cells, decreasing their cytotoxic effect against tumoral cells.3 Interestingly, MSCs from bone marrow (BM-MSC) decrease M1 and increase M2 macrophage polarization in an in vitro coculture system.4 Macrophages and MSCs are present in microenvironment of cervical cancer, however it is unknown if MSCs play a role in macrophage polarization. In the present study, we have evaluated the immunoregulatory capacity of CeCa-MSCs to induce macrophage polarization.MethodsCD14 monocytes were isolated from peripheral blood and cultivated in the absence or presence of MSCs from BM, normal cervix (NCx) and CeCa. Two culture conditions were included, in the presence of induction medium to favors M1 (GM-CSF, LPS and IFNg) or M2 (M-CSF, IL-4 and IL-13) macrophage polarization. M1 (HLA-DR, CD80, CD86 and IFNg) or M2 (CD14, CD163, CD206, IDO and IL-10) macrophage molecular markers were evaluated by flow cytometry. Finally, we evaluated concentration of IL-10 and TNFa in conditioned medium form all coculture conditions.ResultsWe observed that CeCa-MSCs and BM-MSCs in presence of M1 induction medium, decreased M1 macrophage markers (HLA-II, CD80, CD86 and IFNg), and increase the expression of CD14 (M2 macrophage marker). Interestingly, in presence of M2 induction medium, BM-MSCs and CaCe-MSCs but not CxN-MSC increased CD163, CD206, IDO and IL-10 (M2 macrophage markers). We observed a decreased concentration of TNFa in the supernatant medium from all cocultures with MSCs, but only in presence of CeCa-MSCs, increased IL-10 concentration was detected in such cocultures.ConclusionsIn contrast to NCx-MSCs, CeCa-MSCs similarly to BM-MSCs have in vitro capacity to decrease M1 and increase M2 macrophage phenotype.AcknowledgementsAcknowledgments The authors are indebted to gratefully acknowledge to CONACYT (Grant No. 272793) and IMSS (Grant no. 1731) for support to Juan J. Montesinos research.ReferencesMartinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 2014;6-13.Petrillo M, Zannoni GF, Martinelli E, et al. Polarization of tumor-associated macrophages toward M2 phenotype correlates with poor response to chemoradiation and reduced survival in patients with locally advanced cervical cancer. PLoS One 2015;10: e0136654.Montesinos JJ, Mora-García Mde L, et al. In vitro evidence of the presence of mesenchymal stromal cells in cervical cancer and their role in protecting cancer cells from cytotoxic T cell activity. Stem Cells Dev 2013;22:2508-2519.Vasandan AB, Jahnavi S, Shashank C. Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE 2-dependent mechanism. Sci Rep 2016;6:38308.

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