Skeletal Stem Cells - Engineered Bone: Tissue Engineering Intelligence Unit - page 18

Table 2. Colony forming efficiency, morphology and osteogenic potential
of circulating skeletal stem cells

C F E x 1 0 6 C e l l s # P o l y g o n a l / # o f O s t e o g e n i c

( R a n g e b e t w e e n F i b r o b l a s t i c C o l o n i e s C l o n e s / C l o n e s

A n i m a l S p e c i e s D o n o r s ) ( % P o l y g o n a l ) T r a n s p l a n t e d

Mouse 0.93 5/33 1/8

(0-3.8) (13%)

Rabbit 0.18 6/41 3/19

(0-0.58) (13%)

Guinea Pig 2.7 15/319 2/4*

(1.1-3.9) (5%)

Human Rare 0/2 1/2

(0-0.025) (0%)

*Guinea pig cultures were derived as polyclonal rather than single colony derives strains

TGF- β 1), they synthesized cartilage matrix molecules, including collagen type II, VI, and chon-
droitin 4-sulfate. 16 These cells were assayed by in vivo transplantation in three-dimensional
alginate gels. Four and 12 weeks post-transplantation, the cells demonstrated significant pro-
duction of cartilage matrix molecules, 16 though no morphologically recognizable cartilage tis-
sue was formed. Finally, using a different transplantation technique, bone tissue was formed in
vivo by transplanted adipose tissue-derived cells (J.H.F. Justesen, personal communication).
These combined data strongly suggest that subcutaneous adipose tissue contains SSCs with at
least quadruple differentiation potential: osteogenic, chondrogenic, adipogenic, and stromal.
It remains to be found, however, whether these putative subcutaneous SSCs represent a distinct
stem cell type or are derived from pericytes from subcutaneous microvessels.
Recently, adherent, clonogenic cells have been isolated from the blood of four mammalian
species: mouse, rabbit, guinea pig and human. 46 Colony forming efficiency (CFE), or the
number of colonies per 10 6 nucleated cells, varied significantly between species, as well as from
one individual donor to another (Table 2). In all species except human, colonies of two distinct
types were observed. The majority of the colonies were composed of cells with fibroblastic
morphology, but a variable, small proportion of colonies (Table 2) consisted of cells exhibiting
a distinctive polygonal shape. Cloned strains of both types, however, demonstrated virtually
identical immunophenotype, staining positively for some, but not all markers of fibroblastic,
osteogenic, and smooth muscle cells but negatively for markers of endothelial cells, hematopoi-
etic cells, and monocytes/macrophages (Table 1). The blood-derived adherent cells, therefore,
phenotypically resembled but were distinguishable from BMSCs. Notably, the human
blood-derived adherent cells were negative for the marrow stromal marker, Stro-1, as well as for
endoglin and Muc-18, all of which were expressed in human BMSCs. Both blood-derived
adherent cell and BMSC populations were negative for PPAR γ 2 but contained cells positive
for another adipogenic marker, CEBP α . Upon culture with medium containing rabbit serum,
a known inducer of adipogenesis, 14 blood-derived adherent cells from all species underwent
adipogenic differentiation. 46 In micromass cultures in the presence of chondrogenic supple-
ments, 40 a proportion of guinea pig clonal and polyclonal strains of blood-derived adherent
cells demonstrated vigorous cartilage formation (Kuznetsov and Gehron Robey, unpublished
observation).
Clonal strains of blood-derived adherent cells from mouse, rabbit and human cultures and
polyclonal strains from guinea pig cultures were transplanted within HA/TCP powder vehicles
into the subcutis of immunocompromised mice to determine the capability of forming bone.
Histology-proven bone was formed by 12-50% of the strains, depending on the animal species