Mesenchymal Stem Cells - Interest Group
Characteristics:

Human MSCs (hMSCs) are typically isolated from
the mononuclear layer of the bone marrow after
separation by density gradient centrifugation. The
mononuclear cells are cultured in medium with 10%
fetal calf serum, and the MSCs adhere to the tissue
culture plastic. Some hematopoietic cells also
adhere, but over time in culture these are washed
away, leaving adherent, fibroblast-like cells. After an
initial lag phase, the cells divide rapidly, with
population doubling time depending on the donor
and the initial plating density.
MSCs and MSC-like cells have now been isolated from various sites other than the bone marrow,
including adipose tissue, amniotic fluid, periosteum, and fetal tissues, and show phenotypic
heterogeneity. MSC-like cells have been isolated from pathological tissues such as the rheumatoid
arthritic joint, and these cells express bone morphogenetic protein receptors. Indeed, it has been
suggested that cells with mesenchymal stem characteristics reside in virtually all postnatal organs
and tissues. MSCs have been isolated and cultured from many other species including mice, rats,
cats, dogs, rabbits, pigs, and baboons, albeit with varying success, as it can be difficult to remove
contaminating hematopoietic cells from species such as mice. Nevertheless, enrichment for some
species' MSCs can be achieved by expansion and passaging in deprivational medium to eliminate
contamination. The resulting cultures are still morphologically heterogeneous, containing cells
ranging from narrow spindle-shaped cells to large polygonal cells and, in confluent cultures, some
slightly cuboidal cells.

Phenotypically, MSCs express a number of markers, none of which, unfortunately, are specific to
MSCs. It is generally agreed that adult human MSCs do not express the hematopoietic markers
CD45, CD34, CD14, or CD11. They also do not express the costimulatory molecules CD80, CD86,
or CD40 or the adhesion molecules CD31 (platelet/endothelial cell adhesion molecule [PECAM]-1),
CD18 (leukocyte function-associated antigen-1 [LFA-1]), or CD56 (neuronal cell adhesion molecule-
1), but they can express CD105 (SH2), CD73 (SH3/4), CD44, CD90 (Thy-1), CD71, and Stro-1 as
well as the adhesion molecules CD106 (vascular cell adhesion molecule [VCAM]-1), CD166
(activated leukocyte cell adhesion molecule [ALCAM]), intercellular adhesion molecule (ICAM)-1,
and CD29.

There are several reports that describe the isolation of both human and rodent MSCs using antibody
selection based on the phenotype of MSCs. Some have used a method of negative selection to
enrich for MSCs, whereby cells from the hematopoietic lineage are removed; others have used
antibodies to positively select for MSCs.

MSCs from other species do not express all the same molecules as those on human cells; for
example, although human and rat MSCs have been shown to be CD34–, some papers report
variable expression of CD34 on murine MSCs [22]. It is generally accepted that all MSCs are devoid
of the hematopoietic marker CD45 and the endothelial cell marker CD31. However, it is important
to note that differences in cell surface expression of many markers may be influenced by factors
secreted by accessory cells in the initial passages, and the in vitro expression of some markers by
MSCs does not always correlate with their expression patterns in vivo.

There is also variable expression of many of the markers mentioned due to variation in tissue
source, the method of isolation and culture, and species differences. For example, human adipose
tissue is a source of multipotent stem cells called processed lipoaspirate (PLA) cells which, like bone
marrow MSCs, can differentiate down several mesenchymal lineages in vitro. However, there are
some differences in the expressions of particular markers: CD49d is expressed on PLA cells but not
MSCs, and CD106 is expressed on MSCs but not PLA cells. CD106 on MSCs in bone marrow has
been functionally associated with hematopoiesis, so the lack of CD106 expression on PLA cells is
consistent with localization of these cells to a nonhematopoietic tissue.

Blood-derived mesenchymal precursor cells (BMPCs) have also been described in the blood of
normal individuals, and these express many of the same markers as bone marrow MSCs, as well as
differentiating down the osteoblastic and adipogenic lineages. However, these appear to be a
separate population from fibrocytes, which are mesenchymal precursor cells that circulate in the
blood and can migrate into tissues. Fibrocytes express CD34 and CD45 and appear to differentiate
into myofibroblasts, whereas BMPCs are reported to be CD34 negative.

Mesenchymal stem cells have also been isolated from human first- and second-trimester fetal blood,
liver, spleen, and bone marrow. Although phenotypically similar, these culture-expanded MSCs
exhibited heterogeneity in differentiation potential, which related to the tissue source. Taken
together, these examples illustrate that mesenchymal precursor cells are phenotypically
heterogeneous, and the relationship between traditional bone marrow-derived MSCs and these
other MSC-like populations remains to be fully clarified.

Adult human MSCs are reported to express intermediate levels of major histocompatibility complex
(MHC) class I but do not express human leukocyte antigen (HLA) class II antigens on the cell
surface. The expression of HLA class I on fetal hMSCs is lower [28]. Le Blanc and colleagues did
detect HLA class II by Western blot on lysates of unstimulated adult hMSCs, suggesting intracellular
deposits of the antigen [18], and found that cell-surface expression can be induced by treatment of
the cells with interferon-{gamma} for 1 or 2 days. Unlike adult hMSCs, human fetal liver-derived
hMSCs have no MHC class II intracellularly or on the cell surface, suggesting that MHC antigen
expression by hMSCs changes from fetal to adult life.


References:

  • Colter DC, Class R, DiGirolamo CM et al. Rapid expansion of recycling stem cells in cultures
    of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;97:3213–
    3218.

  • Campagnoli C, Roberts IA, Kumar S et al. Identification of mesenchymal stem/progenitor
    cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98:2396–2402.

  • In't Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Amniotic fluid as a novel source
    of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548–1549.

  • Nakahara H, Dennis JE, Bruder SP et al. In vitro differentiation of bone and hypertrophic
    cartilage from periosteal-derived cells. Exp Cell Res 1991;195:492–503.

More References...
Fluorescent murine mesenchymal stem cells in
culture transfected (modified) with a green
fluorescent protein. [Antonio Uccelli, MD, Dept.
of Neurosciences, University of Genoa, Italy.]