Clonogenic analysis reveals reserve stem cells in postnatal mammals: I. Pluripotent mesenchymal stem cells

Abstract

Clonal populations of lineage-uncommitted pluripotent mesenchymal stem cells have been identified in prenatal avians and rodents. These cells reside in the connective tissue matrices of many organs and tissues. They demonstrate extended capabilities for selfrenewal and the ability to differentiate into multiple separate tissues within the mesodermal germ line. This study was designed to determine whether such cells are present in the connective tissues of postnatal mammals. This report describes a cell clone derived by isolation from postnatal rat connective tissues, cryopreservation, extended propagation, and serial dilution clonogenic analysis. In the undifferentiated state, this clone demonstrates a high nuclear-to-cytoplasmic ratio and extended capacity for self-renewal. Subsequent morphological, histochemical, and immunochemical analysis after the induction of differentiation revealed phenotypic markers characteristic of multiple cell types of mesodermal origin, such as skeletal muscle, smooth muscle, fat cells, cartilage, and bone. These results indicate that this clone consists of pluripotent mesenchymal stem cells. This report demonstrates that clonal populations of reserve stem cells are present in mammals after birth. Potential roles for such cells in the maintenance, repair, and regeneration of mesodermal tissues are discussed.

Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic‐like stem cells

“…Postnatal Sprague-Dawley rats were euthanized using CO 2 inhalation, and the fleshy muscle bellies of the thigh and leg musculature were processed for stem cell isolation, cultivation, and cryopreservation (Young et al, 1991(Young et al, , 1998a(Young et al, , 2001aYoung, 2000.…”

“…Previous cloning studies with prenatal chicks , prenatal mice (Rogers et al, 1995;Young et al, 1998a), and postnatal rats (Young et al, 2001a) revealed that repetitive singlecell clonogenic analysis could be achieved if individual cells were grown in medium preconditioned by highly proliferating cells of the same parental line. Therefore, the culture medium from stem cells undergoing postconfluence log-phase expansion was pooled, processed, and stored at -70 to 80°C.…”

“…Previous cloning studies in prenatal mice (Rogers et al, 1995;Young et al, 1998b) and postnatal rats (Young et al, 2001a) revealed that a higher efficacy of cloning could be achieved if cells were cultivated past 50 population doublings prior to cloning. When such cells were incubated with insulin, less than 1% of the cells displayed phenotypic markers for differentiated cells of the various mesodermal tissue lineages (Young, 2000.…”

“…Frozen cells were thawed, grown past confluence, harvested, and cell viability was determined. They were then diluted to clonal density (one cell per 5 l) with cloning medium and plated as described (Young et al, 1998b(Young et al, , 2001a. The process of seeding at single-cell clonal density, propagation past confluence, culture selection (i.e., retention of a stellate morphology, loss of contact inhibition, and growth past confluence), harvest, propagation past confluence in six-well plates, culture selection, harvest, and cryopreservation was repeated two additional times after initial cloning to ensure that each clone was derived from a single cell.…”

“…The process of seeding at single-cell clonal density, propagation past confluence, culture selection (i.e., retention of a stellate morphology, loss of contact inhibition, and growth past confluence), harvest, propagation past confluence in six-well plates, culture selection, harvest, and cryopreservation was repeated two additional times after initial cloning to ensure that each clone was derived from a single cell. The resultant clones were propagated, harvested, and cryopreserved (Young et al, , 1998b(Young et al, , 2001aRogers et al, 1995). Each round of cloning resulted in approximately 20 population doublings.…”