In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4739-4739
Author(s):  
Cristina Castilla-LLorente ◽  
Mineo Iwata ◽  
Marco Mielcarek ◽  
V. Kraig Abrams ◽  
Billanna Hwang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lymph Nodes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Blood Mononuclear Cells ◽  
Post Infusion

Abstract Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Δημιουργία, in vitro πολλαπλασιασμός και διαφοροποίηση ανθρώπινων επαγόμενων πολυδύναμων βλαστοκυττάρων

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.

scholarly journals Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1580
Author(s):  
Yvonne Roger ◽  
Laura Burmeister ◽  
Anika Hamm ◽  
Kirsten Elger ◽  
Oliver Dittrich-Breiholz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Population Doubling ◽  
Pcr Analysis ◽  
Inhibitory Influence ◽  
In Vitro Differentiation ◽  
Cell Surface Antigens

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages—a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

scholarly journals Dose and duration of interferon γ pre-licensing interact with donor characteristics to influence the expression and function of indoleamine-2,3-dioxygenase in mesenchymal stromal cells

2020 ◽  
Vol 17 (167) ◽  
pp. 20190815
Author(s):  
Devlin T. Boyt ◽  
Lauren K. Boland ◽  
Anthony J. Burand ◽  
Alex J. Brown ◽  
James A. Ankrum
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Inflammatory Diseases ◽  
Interferon Γ ◽  
Relative Role ◽  
Peripheral Blood Mononuclear ◽  
High Performing ◽  
Indoleamine 2,3 Dioxygenase

Human mesenchymal stromal cells (MSCs) are a leading cell therapy candidate for the treatment of immune and inflammatory diseases due to their potent regulation of immune cells. MSC expression of indoleamine-2,3-dioxygenase (IDO) upon interferon γ (IFNγ) exposure has been proposed as both a sentinel marker and key mediator of MSC immunomodulatory potency. Rather than wait for in vivo exposure to cytokines, MSCs can be pre-licensed during manufacturing to enhance IDO expression. In this study, we systematically examine the relative role that the dose of IFNγ, the duration of pre-licensing and the donor of origin play in dictating MSC production of functional IDO. We find that across three human MSC donors, MSCs increase their expression of IDO in response to both increased dose of IFNγ and duration of pre-licensing. However, with extended pre-licensing, the expression of IDO no longer predicts MSCs ability to suppress activated peripheral blood mononuclear cells. In addition, pre-licensing dose and duration are revealed to be minor modifiers of MSCs inherent potency, and thus cannot be manipulated to boost poor donors to the levels of high-performing donors. Thus, the dose and duration of pre-licensing should be tailored to optimize performance of specific donors and an emphasis on donor selection is needed to realize significant benefits of pre-licensing.

Human CB-Derived Cells Give Rise to Insulin-Producing-Cells in Xenogeneic Hosts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3603-3603
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Noriaki Kawano ◽  
Hua Zhang ◽  
Yuan Kong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Chromosome ◽  
Human Insulin ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Insulin Producing Cells

Abstract (Purpose) We examine the capacity of human cord blood (CB)-derived cells to generate insulin-producing cells and other lineages of cells in pancreatic tissue in vivo. (Method) Ten millions of human CB-derived T-cell-depleted mononuclear cells were intravenously transplanted into conditioned newborn NOD/SCID/b2-microglobulinnull mice with or without chemical injury by an intraperitoneal injection of streptozotocin (STZ) at dose of 100 mg/g body weight. At 1–3 months post-transplantation, pancreatic tissues of the recipient mice were analyzed for the presence of human CB-derived cells by performing immunofluorescence study (insulin, amylase, or CD45) and FISH analysis for human chromosomes on the same specimens. RNA was isolated from pancreatic tissues of recipient mice, and RT-PCR analysis using human insulin specific primer was performed to examine human insulin at RNA level. Finally, double FISH analysis for human- and murine chromosomes was performed to get an insight into the mechanism for the generation of human CB-derived insulin-producing cells in vivo. (Results) At 1–3 months post-transplantation, human CB-derived T-cell-depleted mononuclear cells gave rise to both myeloid and lymphoid progeny (CD33+, CD19+, and CD3+ cells) in bone marrow and peripheral blood of the recipient mice. In recipient pancreatic tissues, human CB-derived cells were identified inside and outside islets. Outside pancreatic islets, the vast majority of human chromosome+ cells were CD45+ hematopoietic cells, while human chromosome+ amylase+ acinar cells were also identified. Inside islets, human chromosome+ cells accounted for 1.01 +/− 0.73 % (n=6) without STZ treatment. Among them, human CB-derived insulin-producing cells were identified at a frequency of 0.65 +/− 0.64 % (n=6) of total insulin+ cells in xenogeneic hosts. RT-PCR analysis demonstrated the presence of human insulin, whose sequence was fully identical to that of already-known human insulin cDNA. Chemical injury with STZ treatment led to the significant destruction of islet tissue and reduction of cell numbers in islets. In STZ-treated recipient mice, however, human insulin-producing cells were identified at a frequency of 0.23 +/− 0.27 % (n=4) in islets, which was lower than the mice without STZ treatment. Finally, double FISH analyses using species-specific probes demonstrated the presence of human chromosome+ murine chromosome+ insulin-producing cells and human chromosome+ murine chromosome- insulin-producing cells in recipient islets. (Conclusion) It is concluded that human CB cells contain the progenitor cells to generate the insulin-producing cells in vivo. The mechanism of CB-derived insulin-producing cells includes both fusion-dependent and independent mechanisms. Although the capacity of CB-derived cells needs to be compared with other stem cell sources such as tissue stem cells or embryonic stem cells, the present study suggests the possibility of CB cells as new source for future regenerative medicine for diabetes mellitus.

scholarly journals Mesenchymal Stromal Cells Prevent Allostimulation In Vivo and Control Checkpoints of Th1 Priming: Migration of Human DC to Lymph Nodes and NK Cell Activation

Stem Cells ◽  
2015 ◽  
Vol 33 (10) ◽  
pp. 3087-3099 ◽  
Author(s):  
C. Consentius ◽  
L. Akyüz ◽  
J. A. Schmidt-Lucke ◽  
C. Tschöpe ◽  
L. Pinzur ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
And Control
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