Appendix J. 
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Culture of MAPC is, however, technically demanding. Major 
factors that play a role m successful maintenance of MAPC include 
cell density, CO 2 concentration and pH of the medium, lot of fetal calf 
serum that is used, and even the type of culture plastic that is used. 
Control of cell density appears to be species specific: mouse, rat and 
perhaps cynomologous monkey MAPC need to be maintained at 
densities between 500 and 1,000 cells / cm2, whereas human and 
perhaps dog MAPC need to be maintained between 1,500 and 3,000 
cells/ cm^. The reason why MAPC tend to difierentiate to the default 
MSC Lineage when maintained at higher densities is not known. 
However, for MAPC to have clinical relevance, this will need to be 
overcome. Gene array and proteomics studies are ongoing to identify 
the contact and / or soluble factors that may be responsible for 
causing differentiation when MAPC are maintained at higher 
densities. These very demanding technical skills can however be 
“exported” from the University of Mirmesota as, after training at the 
University of Minnesota, investigators at the University of Tokai. 
Japan (manuscript submitted) and investigators at the University of 
Gent, Belgium have successfully isolated MAPC from human bone 
marrow, and investigators at the University of Navarra, Spain, have 
successfully isolated MAPC from rat bone marrow. 
IN VITRO DIFFERENTIATION POTENTIAL OF MAPC: 
We published last year that human, mouse and rat MAPC 
can be successfully differentiated into typical mesenchymal lineage 
cells, including osteoblasts, chondroblaists, adipocytes and skeletal 
myoblasts In addition, human, mouse and rat MAPC can be 
induced to differentiate into cells with morphological, phenotypic 
and functional characteristics of endothehal cells and 
morphological, phenotypic and functional characteristics of 
hepatocytes ^ 
Neuroectodermal differentiation 
Since then, we have also been able to induce differentiation 
of MAPC from mouse bone marrow into cells with morphological, 
phenotypic and functional characteristics of neuroectodermal cells . 
Differentiation of MAPC to cells with neuroectodermal characteristics 
occurred by initial culture in the presence of basic fibroblast growth 
factor (bFGF) as the sole cytokine, followed by culture with FGF-8b 
and sonic hedgehog (SHH), and then brain derived neurotrophic 
factor (BDNF) Differentiation using these sequential cytokine 
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