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Original article from STEM CELLS Translational Medicine

“An Abundant Perivascular Source of Stem Cells for Bone Tissue Engineering”

The isolation of mesenchymal stem cell (MSC)-like cells for bone formation/regeneration currently relies on their ability to adhere to culture plates and their extended growth in vitro, which carries the risk of adaption and loss of function in vivo. A more efficacious method would be the direct identification and isolation of such cells from primary sources, a strategy which has been previously attempted from the freshly isolated total stromal vascular fraction (SVF) from adipose tissue. However, these initial studies found poor bone formation/regeneration (Muller et al and Cheung et al) perhaps linked to the heterogenous nature of the SVF, which contain many non-mesenchymal cell types. Researchers from the laboratories of Kang Ting, Bruno Péault and Chia Soo previously reported on the MSC-like characteristics of human perivascular stem cells (hPSCs) found in adipose tissue, identifying two subpopulations; CD45-CD146+CD34- pericytes surrounding microvessels and capillaries (Crisan et al 2008 and Crisan et al 2009) and a second distinct CD45-CD146-CD34+adventitial cell type, associated with the outermost connective tissue covering larger blood vessels (Corselli et al). Another previous paper from the authors also suggested that hPSCs outperformed human SVF cells (hSVFs) in osteogenic differentiation and bone formation (James et al). Now, in a follow-up study in Stem Cells Translational Medicine the group extend their studies on hPSCs, identifying them as a highly reproducible and efficacious cell source for bone regeneration (James and Zara et al).

Previous work had defined two sources of primary MSCs in adipose tissue on the basis of CD34 and CD146 (MCAM/MUC18) expression; pericytes (CD45-CD146+CD34-) and adventitial cells (CD45-CD146-CD34) and these results where first confirmed after removing dead cells (DAPI+) and haematopoietic cells (CD45+) from lipoaspirate. From 100ml of lipoaspirate, and average of ~40 million SVF cells were collected; ~20% pericytes and ~24% adventitial cells with cell viability above 70%.  Several factors which could affect the yield of these cells where then investigated thoroughly. Cold storage time led to a small non-significant reduction in total SVF yield and CD45+ cell number at 72 hours and 24 hours respectively.   Adventitial cells also showed a slight decrease (non-significant) in yield with time and, interestingly, pericytes showed an increase in yield with time, rising from 14.2% for immediately processed samples to 28.3% for cells processed at 72 hours. Cell yield as a mode of patient age (in decades) was next analyzed, however, no significant change in yield, viability, or identity was observed with patient age. However, when analyzing correlations to gender, it was discovered that SVF cell yield was reduced in males although the percentage of adventitial cells was increased. No differences were noted for the prevalence of CD45+ cells or pericytes, and additionally, the onset of menopause in females had no statistically significant effects on any cell parameters of yield, viability, or identity. Finally, increasing BMI was linked to a reduced SVF yield, increasing frequency of CD45+ cells and reduced frequency of pericytes, although these were all non-significant.

Next, using a mouse critical sized calvarial (skull bone) defect model, the usefulness of the hPSCs (pericytes and adventitial cells) were analysed for bone healing ability. In a previous study the group had shown that hPSCs had increased in vivo bone formation potential in comparison with patient-matched hSVFs in an intramuscular (ectopic) bone model (James et al). The model in this study entailed the insertion of a hydroxyapatite-coated PLGA (poly (lactic-co-glycolic acid)) scaffold seeded with hPSCs or unsorted hSVFs from a matched patient into a 3mm full thickness parietal bone defect. Excitingly, robust bone growth was observed for the hPSC-scaffolds at 8 weeks but not the hSVF-scaffolds which only showed scattered bone growth, as observed by high-resolution live microcomputed tomography scanning and 3D reconstructions. Indeed, hPSC-scaffolds led to 61.5% and 69% healing at 4 and 6 weeks, significantly higher than the hSVF-scaffold, a naked scaffold or no scaffold at all. Further analysis of bone markers (OPN (intermediate marker of osteogenesis) and OCN (late marker of osteogenesis)) by immunohistochemistry only found OPN+ bone-lining osteoblasts and OPN+ osteocytes within the defect site for the hPSC-scaffold. OCN exhibited a more scattered distribution in these assays although OCN was more predominant in the hPSC-scaffold treated mice. Additionally, the growth factors BMP2 and VEGF intensely marked areas only within the hPSC-scaffold, and not the hSVF-scaffold or the naked scaffold.   Finally, the persistence of human cells was analysed, finding that while both hSVF and hPSCs persisted at 8 weeks post-operation, this was more obvious for the hPSC-scaffold, indicative of greater survival or proliferation.

Overall this suggests that purified hPSCs from SVF represents an attractive and plentiful source of cells for therapeutic strategies towards bone defects. They are easily purified, their yield is not affected by any of the parameters studied and they cope well with cold-storage. The authors suggest that given their research, an estimated 200ml of lipoaspirate would be sufficient for the clinical application of hPSCs, an amount which could easily be collected from all but the thinnest of patients, without the need for in vitro culture. The authors also note that these cells are already a potentially approvable stem cell technology as regulated by the U.S. Food and Drug Administration (FDA). hPSCs have the ability not only to generate bone, but also skeletal muscle (Park et al), lung (Montemurro et al) and myocardium (Crisan et al 2009) and a  fuller characterisation of hPSCs may be the final step before their clinical use.

 

References

Cheung WK et al.
Osteogenic comparison of expanded and uncultured adipose stromal cells.
Cytotherapy 2010;12:554 –562.

Corselli M et al.
The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells.
Stem Cells Dev 2012;21:1299 –1308.

Crisan M et al.
A perivascular origin for mesenchymal stem cells in multiple human organs.
Cell Stem Cell 2008; 3:301–313

Crisan M et al.
Perivascular multipotent progenitor cells in human organs.
Ann NY Acad Sci 2009;1176:118– 123.

James AW and Zara JN et al.
An Abundant Perivascular Source of Stem Cells for Bone Tissue Engineering.
Stem Cells Translational Medicine. 2012;1:673-684.

James AW et al.
Perivascular stem cells: A prospectively purified mesenchymal stem cell population for bone tissue engineering.
Stem Cells Translational Medicine. 2012;1:510–519.

Montemurro T et al.
Differentiation and migration properties of human foetal umbilical cord perivascular cells: Potential for lung repair.
J Cell Mol Med 2011;15:796–808.

Muller AM et al.
Towards an intraoperative engineering of osteogenic and vasculogenic grafts from the stromal vascular fraction of human adipose tissue.
Eur Cell Mater 2010;19:127–135.

Park TS et al.
Placental perivascular cells for human muscle regeneration.
Stem Cells Dev 2011;20:451–463.

 

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.