Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation

Abstract

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

Mesenchymal stem cells from cryopreserved human umbilical cord blood

“…On the next confluence, the cells were replated at 1:5 dilution under the same culture conditions. BM-derived MNCs were cultured as described by Bruder et al [8] to obtain MSCs.…”

“…These cells are referred to as mesenchymal stem cells (MSCs), since they are known to have capacity of proliferation and differentiation into the mesenchymal lineage. Their mesenchymal differentiation potential is retained even after repeated subcultivation in vitro [8,9]. Though BM has been represented as the main available source of MSCs [10,11], the use of bone marrow-derived cells is not always acceptable owing to the high degree of viral exposure and the significant decrease in the cell number and the proliferative/differentiation capacity along with age.…”

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

“…To assess mineralization, deposit calcium in cultures was stained with silver nitrate (Sigma) by the method of von Kossa [21].…”

Updates on stem cells and their applications in regenerative medicine

“…A heterogonous population of cells was retrieved, with some adherent cells exhibiting a spindle shape; also, they multiplied rapidly in vitro, forming distinct colonies called colony-forming unit fibroblasts (CFU-Fs). Eventually, these cells were successfully isolated from several mammals, including humans (Ouyang et al, 2004;Bruder et al, 1997). In addition, CFU-Fs were found to have multipotent potential, thus implying that they have stem cell properties (Owen, 1988;Bianco et al, 2001).…”