In vitro expansion and differentiation of fresh and revitalized adult canine bone marrow-derived and adipose tissue-derived stromal cells

The objective of this study was to determine the tissue density, in vitro expansion and differentiation of canine adipose tissue-derived (ASC) and bone marrow-derived (BMSC) stromal cells. Primary (P0) and cell passages 1–6 (P1–6) cell doubling numbers (CD) and doubling times (DT) were determined in fresh cells. The P0, P3, and P6 adipogenic (CFU-Ad), osteogenic (CFU-Ob), and fibroblastic (CFU-F) colony forming unit frequencies, lineage specific mRNA levels in differentiated P3 cells and composition of P3 and P6 chondrogenic pellets were assessed in cryogenically preserved cells. Cell yields from bone marrow were significantly higher than adipose tissue. Overall ASC and BMSC CDs and DTs and P3 and P6 CFU-F, CFU-Ad, and CFU-Ob were comparable. The P0 BMSC CFU-Ob was significantly higher than ASC. Lineage specific mRNA levels were higher in differentiated versus control cells, but similar between cell types. Protein was significantly greater in P3 versus P6 ASC chondrogenic pellets. Based on these findings, fresh and revitalized canine ASCs are viable alternatives to BMSCs for stromal cell applications

Adipose tissue as a stem cell source for musculoskeletal regeneration

Adipose tissue is an abundant, easily accessible, and reproducible cell source for musculo-skeletal regenerative medicine applications. Initial derivation steps yield a heterogeneous population of cells collectively termed the stromal vascular fraction (SVF), which consist of endothelial cells, immune cells, pericytes, and pre-adipocytes. Subsequent selection of an adherent cell subset from the SVF results in a relatively homogeneous population of adipose-derived stromal/stem cells (ASCs). Mammalian ASCs exhibit the ability to selectively differentiate into chondrogenic, myogenic, and osteogenic lineages in response to inductive stimuli in vitro (when cultured on scaffolds in bioreactors) and in vivo (when implanted in pre-clinical animal models). Unlike hematopoietic cells, ASCs do not elicit a robust lymphocyte reaction and instead generate and release immunosuppressive factors, such as prostaglandin E2. These unique immunomodulatory features suggest that both allogeneic and autologous ASCs will engraft successfully following application for tissue regeneration purposes. The differentiation and expansion potential of ASCs can be modified by growth factors like bone morphogenetic protein 6, bio-inductive scaffolds, and bioreactors providing environmental control and biophysical stimulation. Gene therapy approaches using lentiviral transduction can also be used to direct differentiation of ASCs along particular lineage pathways. We discuss here the utility of ASCs for musculo-skeletal tissue repair and some of the technologies that can be implemented to unlock the full regenerative potential of these highly valuable cells

Impaired expansion and multipotentiality of adult stromal cells in a rat chronic alcohol abuse model

It is well established that bone maintenance and healing is compromised in alcoholics. Adult mesenchymal stromal cells (MSCs) in bone marrow (BMSCs) and adipose tissue (ASCs) likely contribute to bone homeostasis and formation. Direct and indirect alcohol exposure inhibits osteoprogenitor cell function through a variety of proposed mechanisms. The goal of this study was to characterize the effects of chronic alcohol ingestion on the native number and in vitro growth characteristics and multipotentiality of adult BMSCs and ASCs in a rat model. Adult male Sprague-Dawley rats received a liquid diet containing 36% ethanol (EtOH) or an isocaloric substitution of dextramaltose (control). After 4, 8, or 12 weeks of the diet, ASCs were harvested from epididymal adipose tissue and BMSCs from femoral and tibial bone marrow. Cell doublings per day (CDs) and doubling times (DT) were determined for primary cells (P0) and cell passages 1 through 6 (P1-6). Fibroblastic, (CFU-F), adipogenic (CFU-Ad) and osteogenic (CFU-Ob) colony forming unit frequencies were assessed for P0, P3, and P6. The CDs and DTs were lower and higher, respectively, for ASCs and BMSCs harvested from EtOH versus control rats at all time points. The CFU-F, CFU-Ad and CFU-Ob were significantly higher in ASCs harvested from control versus EtOH rats for P0, P3, and P6 at all times. Both CFU-Ad and CFU-Ob were significantly higher in P0 BMSCs harvested from control versus EtOH rats after 12 weeks of the diet. The CFU-Ob for P3 BMSCs from control rats was significantly higher than those from ETOH rats after 8 and 12 weeks on the diet. All three CFU frequencies in ASCs from EtOH rats tended to decrease with increasing diet duration. The ASC cell and colony morphology was different between control and EtOH cohorts in culture. These results emphasize the significant detrimental effects of chronic alcohol ingestion on the in vitro expansion and multipotentiality of adult MSCs. Maintenance of the effects through multiple cell passages in vitro suggests cells may be permanently compromised