Chapter 2
Mesenchymal Stem Cells from Adult Bone
Marrow
Mark F. Pittenger
Abstract Mesenchymal stem cells (MSCs), sometimes referred to as marrow stro-
mal cells or multipotential stromal cells, represent a class of adult progenitor cells
capable of differentiation to several mesenchymal lineages. They can be isolated
from many tissues although bone marrow has been used most often. The MSCs
may prove useful for repair and regeneration of a variety of mesenchymal tissues
such as bone, cartilage, muscle, marrow stroma, and the cells produce useful
growth factors and cytokines that may help repair additional tissues. There is also
evidence for their differentiation to nonmesenchymal lineages, but that work will
not be considered here. This chapter will provide the researcher with some back-
ground, and then provide details on MSC isolation, expansion and multilineage
differentiation. These are the beginning steps toward formulating tissue repair strat-
egies. The methods provided here have been used in many laboratories around the
world and the reader can begin by following the methods presented here, and then
test other methods if these prove unsatisfactory for your intended purpose.
Keywords Mesenchymal stem cells (MSCs); direct plating isolation; density
gradient isolation, lineage differentiation protocols; chondrogenic; adipogenic;
osteogenic.
1 Introduction
Tissue healing takes place more rapidly in children than in adults and this is likely
owing to a number of factors. One of those factors appears to be the abundance of
stem and progenitor cells in the developing tissues of the child. As we reach adult-
hood, these cells are not so necessary for tissue growth and appear to diminish over
time, perhaps some differentiating to adult cell types whereas some are retained as
resident tissue stem cells. Over the years, the number of tissue resident stem cells
further diminishes as they are called on for normal tissue repair and maintenance,
27
From: Methods in Molecular Biology, vol. 449, Mesenchymal Stem Cells: Methods and Protocols
Edited by D.J. Prockop, D.G. Phinney, and B.A. Bunnell ' Humana Press, Totowa, NJ

28 M.F. Pittenger
and normal cellular senescence. That said, there appear to be small numbers of progenitor
or stem cells that can be isolated from many tissues at all stages of life. These cells
appear to afford a wonderful opportunity, indeed, a responsibility, to understand
important aspects of human biology involving tissue repair and regeneration.
One of these adult stem cells that can be found in several tissues throughout life
and that can be isolated and propagated in culture was termed the mesenchymal stem
cell or MSC (1,2) by Arnold Caplan of Case Western University. A key element in
the acceptance of MSCs as a potential cellular therapeutic was the early demonstra-
tion of safety in humans by Drs. Hillard Lazarus and Stan Gerson, hematological
oncologists, who first tested MSCs as support cells during hematopoietic stem cell
(HSC) transplantations (3). Work began on the isolation of MSCs and examination
of their multipotential nature in 1994, and the multilineage in vitro differentiation of
these cells was demonstrated at the 1996 annual meeting of the American Society
for Cell Biology. This work led to a key paper in the stem cell field published in
Science in 1999 demonstrating multilineage differentiation of clonal populations of
human MSCs that has now been cited in over 2,500 publications (4).
The first descriptions of fibroblastic cells that could be isolated and grown from
bone marrow samples, that retained the ability to differentiate to bone tissue was
presented by Dr. Alexander Friedenstein of the Gamalaya Institute in Moscow in
the 1960s, using guinea pig bone marrow as the source (5–7). When these cells
were culture expanded ex vivo, and then placed in capsules under the skin of a
recipient syngeneic animal, new bone and cartilage tissue was identified when his-
tology was performed. Although the same type of cell, or a close homologue of it,
can be found in many tissues, including adipose (8–11), the endosteal surface of
bone and bone itself, bone marrow has proven to be a reproducible and convenient
source of these cells from all species tested. MSCs have been isolated from mouse
(12–16), rat (17–21), guinea pig (5,6) rabbit (22–24), dog (25,26), goat (27), pig
(28–31), nonhuman primates (32–35), and man (1–4,36–40).
MSCs secrete growth factors and cytokines that have autocrine and paracrine activi-
ties. The MSCs produce vascular endothelial growth factor (VEGF), stem cell factor
(SCF-1), leukemia inhibitory factor (LIF), granulocyte colony stimulatory factor
(G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage
colony stimulating factor(GM-CSF), interleukins (IL-1, -6,-7, -8, -11, -14, and -15),
stromal cell-derived factor (SDF-1), Flt-3 ligand, and others (4,36–39). The expression
of these factors may be modulated through interactions with other cell types (48–50).
Some additional interesting and important aspects of MSCs that have come to
light include their homing to sites of tissue injury, particularly ischemic regions of
heart (26,29,43,44) where the MSCs may prevent deleterious remodeling (28,30,31).
MSCs also have the ability to modify immune responses and engraft in allogeneic
recipients, and MSC treatment has been used to clinically treat graft-versus-host
disease (GVHD) (46–51). MSCs are also under evaluation for clinical use in chil-
dren with osteogenesis imperfecta, and glycogen storage diseases (52–54).
Although methods in these areas are not detailed here, clearly, MSCs represent a
new, exciting and potentially powerful paradigm for cellular therapy.
Although a number of research groups investigated MSCs from nonhuman spe-
cies, Arnold Caplan, Steve Haynesworth, and colleagues at Case Western Reserve