Abstract P-41: Contribution of Matrix-bound Vesicles Produced by Mesenchymal Stromal Cells in the Differentiation of Multipotent Stem Cells in vitro

Background: According to the current view on the extracellular matrix (ECM) composition and functions, it includes not only structural proteins and components of cell adhesion, but also various deposited components, including enzymes involved in ECM remodeling, growth factors, and matrix-bound vesicles (MBV). MBV can presumably participate in the formation of a specific microenvironment for stem cells and regulate their differentiation. However, the contribution of MBV to these processes remains poorly understood. In our work, we evaluated the effects of MBV within native ECM produced by mesenchymal stromal cells (MSCs) cultured in cell sheet on multipotent stem cell differentiation. Methods: We isolated MBV from decellularized MSC-produced ECM by treatment with the following enzymes: collagenase, hyaluronidase, or trypsin, and centrifugation on 1000 kDa filters. The nanostructure and relative size in each sample were observed using TEM. The particle size and concentration were also studied with NTA. In addition, the obtained MBV were examined for the presence of key exosome markers using Western blot. Then we investigated the effect of MBV on the formation of capillary-like structures by endothelial cells (in vitro model of angiogenesis) as well as on the differentiation of primary MSCs isolated from human adipose tissue in the adipogenic, osteogenic, and chondrogenic directions. Results: As a result of comparative analysis of isolation protocols, it was shown that all MBV samples had the characteristics of extracellular vesicles (EV), but differed in size and representation of exosomal markers. The MBV isolated from ECM did not stimulate the formation of capillary-like structures by endothelial cells, in contrast to EV secreted by MSCs to the conditioned medium, but maintained the viability of the endothelium. Isolated MBV stimulated osteogenic and adipogenic differentiation of MSCs similar to secreted EV. On the other hand, preincubation of MSCs with MBV leads to reorganization of cell monolayer to spheroid-like structures during chondrogenic differentiation. Conclusion: Here, we developed the protocol of isolation of MBV from ECM that have distinguished characteristics and functional activity.

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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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Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

Frontiers in Immunology ◽

10.3389/fimmu.2017.01991 ◽

2018 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Clémence Roux ◽

Gaëlle Saviane ◽

Jonathan Pini ◽

Nourhène Belaïd ◽

Gihen Dhib ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Regulatory T Cells ◽

Induced Pluripotent Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Pluripotent Stem Cells ◽

Induced Pluripotent

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Δημιουργία, in vitro πολλαπλασιασμός και διαφοροποίηση ανθρώπινων επαγόμενων πολυδύναμων βλαστοκυττάρων

10.12681/eadd/38471 ◽

2016 ◽

Author(s):

Ιωάννα Βαρελά

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Pluripotent Stem Cells ◽

Rt Pcr ◽

Induced Pluripotent

Η ανακάλυψη της μεθόδου του κυτταρικού επαναπρογραμματισμού ανθρώπινων δερματικών ινοβλαστών σε επαγόμενα πολυδύναμα βλαστοκύτταρα (induced pluripotent stem cells, iPSCs) το 2007 άνοιξε το δρόμο για τη μελέτη και την εξατομικευμένη θεραπεία πολλών χρόνιων νόσων. Επιδιώξαμε να δημιουργήσουμε iPS - κυτταρικές σειρές επαναπρογραμματίζοντας μεσεγχυματικά στρωματικά κύτταρα (mesenchymal stromal cells, MSCs) μυελού των οστών, μέσω μιας μεθόδου επαναπρογραμματισμού χωρίς ενσωμάτωση γονιδίων στο γενετικό υλικό των κυττάρων. Δερματικοί ινοβλάστες από φυσιολογικούς δότες και μεσεγχυματικά στρωματικά κύτταρα μυελού των οστών από φυσιολογικό δότη μεταμόσχευσης μυελού των οστών και από ασθενή με β-Μεσογειακή αναιμία (β-ΜΑ) διαμολύνθηκαν, μέσω λιποσωματικών φορέων, με συνθετικά mRNA που κωδικοποιούν τους μεταγραφικούς παράγοντες Oct4, Klf4, Sox2, Lin28, c-Myc. Στη συνέχεια, τα κύτταρα ελέγχθηκαν σε καλλιέργειες για τον σχηματισμό αποικιών πολυδύναμων βλαστοκυττάρων. Οι αποικίες απομονώθηκαν και με συνεχείς ανακαλλιέργειες δημιουργήθηκαν κυτταρικές σειρές, οι οποίες εξετάστηκαν για την πολυδυναμία τους με μεθόδους ανίχνευσης της έκφρασης των μεταγραφικών παραγόντων πολυδυναμίας (κυτταρομετρία ροής, RT-PCR, μελέτη του μεταγραφώματος με RNA μικροσυστοιχίες). Ως θετικός μάρτυρας και μέτρο σύγκρισης χρησιμοποιήθηκε πολύ καλά χαρακτηρισμένη εμβρυονική σειρά πολυδύναμων βλαστοκυττάρων. Οι iPS-κυτταρικές σειρές μελετήθηκαν, επίσης, ως προς τη λειτουργική τους πολυδυναμία με τον έλεγχο της ικανότητας τους να δημιουργούν in vitro εμβρυϊκά σωματίδια και in vivo τερατώματα μετά από υποδόρια εμφύτευση τους σε ανοσοανεπαρκείς ποντικούς, και ως προς τη δυνατότητα διαφοροποίησής τους σε αιμοποιητικά προγονικά κύτταρα. Η γενετική σταθερότητα των κυτταρικών σειρών ελέγχθηκε με DNA μικροσυστοιχίες συγκριτικού γονιδιωματικού υβριδισμού (aCGH). Απομονώθηκαν 3 iPS κυτταρικές σειρές από κάθε δείγμα κυττάρων, οι οποίες εμφανίζουν μεταγράφωμα πανομοιότυπο με εκείνο των πολυδύναμων εμβρυονικών βλαστοκυττάρων και. δημιουργούν εμβρυϊκά σωματίδια in vitro και τερατώματα in vivo, τα οποία αποτελούνται από ιστούς καταγωγής και από τα τρία βλαστικά δέρματα. Τα iPSCs των κυτταρικών σειρών πολλαπλασιάζονται για μεγάλο χρονικό διάστημα χωρίς μορφολογικές ενδείξες διαφοροποίησης. Με τη μέθοδο aCGH, στις iPS κυτταρικές σειρές μετά την 10η ανακαλλιέργεια ανιχνεύθηκαν πολυμορφισμοί στον αριθμό αντιγράφων (CNVs), τα οποία ήταν ελλείμματα μεγέθους περίπου 3 Mb. Η διαφοροποίηση των iPSCs σε αιμοποιητικά προγονικά κύτταρα οδήγησε στην παραγωγή CD34+ κυττάρων σε ποσοστό 8-10% των παραχθέντων κυττάρων με ασθενούς έντασης συνέκφραση του CD45, προσομοιάζοντας στο αιμαγγειακό στελεχιαίο κύτταρο. Στην παρούσα διατριβή παρουσιάζεται, για πρώτη φορά στην Ελλάδα, εξ όσων γνωρίζουμε, η τεχνολογία παραγωγής ανθρώπινων iPSCs με μια ασφαλή και αξιόπιστη μέθοδο. Οι iPSCs-κυτταρικές σειρές μπορεί να χρησιμοποιηθούν στη μελέτη ασθενειών, στον έλεγχο φαρμάκων και στην ανάπτυξη πρωτοκόλλων ιστικής μηχανικής και κυτταρικής θεραπείας.

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In Vitro Characterization of Multipotent Mesenchymal Stromal Cells Isolated from Palatal Subepithelial Tissue Grafts

Microscopy and Microanalysis ◽

10.1017/s143192761201433x ◽

2013 ◽

Vol 19 (2) ◽

pp. 370-380 ◽

Cited By ~ 13

Author(s):

Alexandra Roman ◽

Andrada Şoancă ◽

Adrian Florea ◽

Emőke Páll

Keyword(s):

Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Embryoid Body ◽

Basic Research ◽

Multipotent Mesenchymal Stromal Cells ◽

Positive Staining ◽

Basic Characteristics ◽

Tissue Grafts

AbstractThe aim of this study was to analyze whether the mesenchymal stromal cells (MSCs) isolated from palatal tissue grafts harvested in order to cover gingival recessions have the basic characteristics of stem cells. The palatal tissue cells were processed using a special culture medium that stimulated the development of only undifferentiated cellular lines. Cells at passage 4 were evaluated by flow cytometry to examine the expression of specific surface markers and were tested for multilineage differentiation capacity. These cells collected at passage 4 were also investigated for the capacity to cluster into embryoid body aggregates. Palatal MSCs displayed positive staining for the mesenchymal markers CD29, CD73, CD105, CD 49e, and CD44, but did not express hematopoietic markers CD34/45. The palatal MSCs successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. When seeded in special conditions, palatal MSCs propagated into unattached spheres resembling embryoid body aggregates consisting both of differentiated and undifferentiated cells as revealed at the ultrastructural evaluation. It is concluded that the isolated palatal MSCs fulfilled the basic criteria defining the stem cells. This new source of stem cells characterized here for the first time opens new perspectives on possible applications in basic research and in regenerative medicine.

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In vitro Assessment of the DNA Damage Response in Dental Mesenchymal Stromal Cells Following Low Dose X-ray Exposure

Frontiers in Public Health ◽

10.3389/fpubh.2021.584484 ◽

2021 ◽

Vol 9 ◽

Author(s):

Niels Belmans ◽

Liese Gilles ◽

Jonas Welkenhuysen ◽

Randy Vermeesen ◽

Bjorn Baselet ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cell Cycle Progression ◽

Deciduous Teeth ◽

Low Doses ◽

Cycle Progression ◽

X Ray

Stem cells contained within the dental mesenchymal stromal cell (MSC) population are crucial for tissue homeostasis. Assuring their genomic stability is therefore essential. Exposure of stem cells to ionizing radiation (IR) is potentially detrimental for normal tissue homeostasis. Although it has been established that exposure to high doses of ionizing radiation (IR) has severe adverse effects on MSCs, knowledge about the impact of low doses of IR is lacking. Here we investigated the effect of low doses of X-irradiation with medical imaging beam settings (<0.1 Gray; 900 mGray per hour), in vitro, on pediatric dental mesenchymal stromal cells containing dental pulp stem cells from deciduous teeth, dental follicle progenitor cells and stem cells from the apical papilla. DNA double strand break (DSB) formation and repair kinetics were monitored by immunocytochemistry of γH2AX and 53BP1 as well as cell cycle progression by flow cytometry and cellular senescence by senescence-associated β-galactosidase assay and ELISA. Increased DNA DSB repair foci, after exposure to low doses of X-rays, were measured as early as 30 min post-irradiation. The number of DSBs returned to baseline levels 24 h after irradiation. Cell cycle analysis revealed marginal effects of IR on cell cycle progression, although a slight G2/M phase arrest was seen in dental pulp stromal cells from deciduous teeth 72 h after irradiation. Despite this cell cycle arrest, no radiation-induced senescence was observed. In conclusion, low X-ray IR doses (< 0.1 Gray; 900 mGray per hour), were able to induce significant increases in the number of DNA DSBs repair foci, but cell cycle progression seems to be minimally affected. This highlights the need for more detailed and extensive studies on the effects of exposure to low IR doses on different mesenchymal stromal cells.

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A Xeno-Free Strategy for Derivation of Human Umbilical Vein Endothelial Cells and Wharton’s Jelly Derived Mesenchymal Stromal Cells: A Feasibility Study toward Personal Cell and Vascular Based Therapy

Stem Cells International ◽

10.1155/2020/8832052 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Hataiwan Kunkanjanawan ◽

Tanut Kunkanjanawan ◽

Veerapol Khemarangsan ◽

Rungrueang Yodsheewan ◽

Kasem Theerakittayakorn ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Umbilical Vein ◽

Wharton’S Jelly ◽

Human Umbilical Vein ◽

Wharton's Jelly ◽

Umbilical Vein Endothelial Cells

Coimplantation of endothelial cells (ECs) and mesenchymal stromal cells (MSCs) into the transplantation site could be a feasible option to achieve a sufficient level of graft-host vascularization. To find a suitable source of tissue that provides a large number of high-quality ECs and MSCs suited for future clinical application, we developed a simplified xeno-free strategy for isolation of human umbilical vein endothelial cells (HUVECs) and Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) from the same umbilical cord. We also assessed whether the coculture of HUVECs and WJ-MSCs derived from the same umbilical cord (autogenic cell source) or from different umbilical cords (allogenic cell sources) had an impact on in vitro angiogenic capacity. We found that HUVECs grown in 5 ng/ml epidermal growth factor (EGF) supplemented xeno-free condition showed higher proliferation potential compared to other conditions. HUVECs and WJ-MSCs obtained from this technic show an endothelial lineage (CD31 and von Willebrand factor) and MSC (CD73, CD90, and CD105) immunophenotype characteristic with high purity, respectively. It was also found that only the coculture of HUVEC/WJ-MSC, but not HUVEC or WJ-MSC mono-culture, provides a positive effect on vessel-like structure (VLS) formation, in vitro. Further investigations are needed to clarify the pros and cons of using autogenic or allogenic source of EC/MSC in tissue engineering applications. To the best of our knowledge, this study offers a simple, but reliable, xeno-free strategy to establish ECs and MSCs from the same umbilical cord, a new opportunity to facilitate the development of personal cell-based therapy.

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MT1-MMP Plays a Critical Role in Hematopoiesis by Regulating HIF-Mediated Chemo-/Cytokine Gene Transcription within Niche Cells.

Blood ◽

10.1182/blood.v120.21.2351.2351 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2351-2351

Author(s):

Chiemi Nishida ◽

Kaori Sato-Kusubata ◽

Yoshihiko Tashiro ◽

Ismael Gritli ◽

Aki Sato ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Gene Transcription ◽

Stromal Cells ◽

Stromal Cell ◽

Stem Cell Differentiation ◽

Cell Phenotype ◽

Hematopoietic Stem

Abstract Abstract 2351 Stem cells reside in a physical niche. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, stem cell maintenance and regeneration. Various stem cell niches have been shown to be hypoxic, thereby maintaining the stem cell phenotype of e.g. hematopoietic stem cells (HSCs) or cancer stem cells. The bone marrow (BM) niche is a rich reservoir of tissue-specific pluripotent HSCs. Proteases such as matrix metalloproteinases (MMPs) have been implicated in cell movement, partly due to their proteolytic function, and they have been linked to cellular processes such as cell proliferation and differentiation. The proteolytic function of Membrane-type 1 MMP (MT1-MMP/MMP-14) is essential for angiogenesis, arthritis and tumour growth. Recently, it has been reported that MT1-MMP is highly expressed in HSCs and stromal/niche cells. However the clear function of MT1-MMP in hematopoiesis is not well understood. To reveal the functional consequences of MT1-MMP deficiency for post-natal hematopoiesis in vivo, we have taken advantage of MT1-MMP−/− mice to demonstrate that MT1-MMP deficiency leads to impaired steady state hematopoiesis of all hematopoietic cell lineages. In a search for factors whose deficiency could cause this hematopoietic phenotype, we found not only reduced protein release, but also reduced transcription of the following growth factors/chemokines in MT1-MMP−/− mice: erythropoietin (Epo), stromal cell-derived factor-1 (SDF-1a/CXCL12), interleukin-7 (IL-7) and Kit ligand (KitL, also known as stem cell factor). All of these factors, except for Epo, are typical stromal cell-derived factors. To ensure that impaired gene transcription in vivo was not due to a lower number of stromal cells in vivo, we demonstrated that MT1-MMP knockdown in stromal cells in vitro also reduced transcription of the stromal cell derived factors SDF-1a/CXCL12, IL-7 and KitL. In contrast, overexpression of MT1-MMP in stromal cells enhanced gene transcription of these factors. All genes, whose transcription was altered in vitro and in vivo due to MT1-MMP deficiency, had one thing in common: their gene transcription is regulated by the hypoxia inducible factor-1 (HIF-1) pathway. Further mechanistic studies revealed that MT1-MMP activates the HIF-1 pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration. Disclosures: No relevant conflicts of interest to declare.

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