A Agrawal

A Madeira

BP Dranka

CM Krawczyk

D Weismann

DGA Burton

DV Ziegler

GJW van der Windt

J Carlos Sepúlveda

J Folch

JA Ankrum

JA Berliner

JA Sánchez-Alcázar

JWD Fijter

K Ito

L von Bahr

LC Amado

M Seyerl

M-K Chang

ME Bernardo

MM Bonab

NJ MacIver

P Suárez-Cortés

R Oliveira

RR Makkar

S Blüml

S Dhingra

S Fazel

S Gupta

SK Roy Chowdhury

TA Seimon

V Serbulea

VN Bochkov

Figure S1. Differentiation of MSCs into osteocytes, adipocytes, and chondrocytes. MSCs were induced to differentiate toward osteocytes (a), adipocytes (b), and chondrocytes (c). MSCs (undifferentiated cells, control group) and differentiated MSCs (D-MSC) were stained for osteocalcin and Alizarin Red (osteocyte lineage), FABP4 and Oil Red-O stain (adipocyte lineage), and aggrecan (chondrocyte lineage). The images were taken using Cytation5 (BioTek Instruments) (20Ă magnification). (nâ=â6) (PPTX 2648 kb

Niketa Sareen (3582215)

Glen Sequiera (3582203)

Rakesh Chaudhary (5168186)

Ejlal Abu-El-Rub (5168183)

Subir Chowdhury (5168180)

Vikram Sharma (142773)

Arun Surendran (4503196)

Meenal Moudgil (5168177)

Paul Fernyhough (1942030)

Amir Ravandi (501272)

Sanjiv Dhingra (343819)

The Francis Crick Institute

Additional file 1: of Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

Sareen, Niketa

Sequiera, Glen Lester

Chaudhary, Rakesh

Abu-El-Rub, Ejlal

Chowdhury, Subir Roy

Sharma, Vikram

Surendran, Arun

Moudgil, Meenal

Fernyhough, Paul

Ravandi, Amir

Dhingra, Sanjiv

PEARL (Univ. of Plymouth)

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

Abstract Background Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3–7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation. Methods The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform. Results Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege. Conclusions The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs

Niketa Sareen

Glen Lester Sequiera

Rakesh Chaudhary

Ejlal Abu-El-Rub

Subir Roy Chowdhury

Vikram Sharma

Arun Surendran

Meenal Moudgil

Paul Fernyhough

Amir Ravandi

Sanjiv Dhingra

Directory of Open Access Journals

Stem Cell Research & Therapy

Abstract
              
                Background
                Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3–7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation.
              
              
                Methods
                The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform.
              
              
                Results
                Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege.
              
              
                Conclusions
                The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs

Sequiera, Glen L

Chowdhury, Subir R

MSpace at the University of Manitoba

Crossref

https://figshare.com/articles/Additional_file_1_of_Early_passaging_of_mesenchymal_stem_cells_does_not_instigate_significant_modifications_in_their_immunological_behavior/6215090

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

Abstract

Similar works

Full text

Available Versions

The Francis Crick Institute

PEARL (Univ. of Plymouth)

Directory of Open Access Journals

MSpace at the University of Manitoba

Crossref