Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

Summary

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

Abstract

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Introduction

Human mesenchymal stem cells (MSCs) are somatic progenitors that can differentiate into the paraxial mesodermal lineages of bone, cartilage, and adipose tissue^1-4, as well as to a few extramesodermal lineages⁵. First isolated from the adult bone marrow, these multilineage progenitors have now been found in numerous tissues^6-8 and, unexpectedly, shown to have strong immunomodulatory properties that appear highly amenable to clinical application^9-12. Detailed mechanisms involved in the immunomodulatory effects are actively being investigated for effective application on specific disease entities. One of the most straightforwar....

Protocol

Patient informed consent as approved by the institutional review board must be obtained for use of human cells.

1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

1. Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette.
   1. Dilute cells with 25 ml of phosphate-buffered saline (PBS).
2. Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting the tube, very slowly and carefully add in 25 ml of the diluted cell suspension over the density gradient so that there is no mixing of the whole blood with the density gradient, i.e

Results

Figure 1 denotes the overall schema of the experiment, and Figure 2 demonstrates the appearance of the various cell culture conditions as visualized by phase-contrast inverted microscopy. MSCs are adherent cells with a fibroblastic, spindle-shaped morphology, whereas PBMCs and leukocytes are small round non-adherent cells. These two morphologically different cell types can be clearly seen in the co-culture. At the end of the assay, when the PBMCs (or leuckotyes) are aspirated for flow cytometric analyses, ev.......

Discussion

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells^18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application.

One of the most critical technical aspects for success in these assays is having adequate PBMC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque PLUS	GE Healthcare	71-7167-00 AG	Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)
Vibrant CFDA-SE Cell Tracer Kit (CFSE)	Life Technologies	V12883	Cellular label for detection of cell division
Phytoagglutinin (PHA)	Sigma	L8902	Activation of human PBMCs
Dynabeads Human T-Activator CD3/28	Life Technologies	111.32D	Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells, etc.
autoMACS™ Separator	Miltenyi Biotec	autoMACS™ Separator	Magnetic based cell separator
autoMACS® Columns	Miltenyi Biotec	130-021-101	separation columns
CD14 microbeads, human	Miltenyi Biotec	130-050-201	For positive selection of CD14+ human monocytes and macrophages from PBMCs
CD4 microbeads, human	Miltenyi Biotec	130-045-101	For positive selection of CD4+ human T lymphocytes from PBMCs
RPMI 1640 Medium	Life Technologies	11875	Human PBMC/leukocyte culture medium
DMEM, Low glucose, pyruvate	Life Technologies	11885	Human mesenchymal stem cell (MSC) culture medium
L-glutamine	Life Technologies	25030-081	Supplementation for MSC complete medium
Penicillin/Streptomycin	Life Technologies	15070-063	Supplementation for PBMC/leukocyte and MSC complete medium
Fetal bovine serum (FBS)	1) Hyclone, for MSC culture                   2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)	1) SH30070.03M 2) 10091-148	Pre-test lots for support of MSC in vitro culture
24-well cell culture plate	Corning	COR3524	Co-culture plate
50 ml centrifuge tube	Corning	430291	Isolation PBMCs from whole blood by Ficoll-Paque PLUS
15 ml centrifuge tube	Corning	430766	Collection of the labeled and unlabeled cell fractions
Round-bottom tubes	BD Falcon	352008	Collection of cells for flow cytometric analysis