Adipose regenerative cells

Adipose tissue contains a high concentration of regenerative cells, including mesenchymal stem cells (MSCs). MSCs are multipotent cells that have the ability to differentiate to mesodermal lineages such as adipocytes, osteocytes, and chondrocytes. The use of MSCs has gained significant interest in many medical fields particularly for regenerative medicine and tissue engineering. There have been promising results in the therapy of myocardial infarction, liver cirrhosis, cornea damage and other disorders where tissue regeneration is crucial.¹²¹ For many decades, bone marrow has been used as the main source of MSCs.

2.3.3.1 Adipose tissue-derived mesenchymal stem cells (AT-MSC)

Presently, an increasing interest is devoted to MSC isolated from adipose tissue (AT-MSC). ^122,123 Adipose tissue, as a MSCs source, presents several advantages in comparison to bone marrow:

(i) adipose tissue is easier to harvest, (ii) it is widely available, (iii) MSC concentration is ~1000x more and (iv) AT-MSC grows faster in culture. ^123,124 Zuk et al. ¹²³ were the first to demonstrate that AT-MSCs are similar to bone marrow-derived mesenchymal stem cells (BM-MSCs) but not identical. Each of them has specific genomic and proteomic markers. As BM-MSCs, AT-MSCs are multipotent but more easily oriented toward an adipose phenotype, whereas BM-MSCs into osteoblasts and chondrocytes.¹²⁵

AT-MSCs are mostly pericytes¹²⁶ that could be in perivascular position and be obtained from the stromal vascular fraction (SVF) of adipose tissue.^123,127 SVF can be extracted from adipose tissue simply by collagenase and centrifugation. SVF is strongly heterogeneous and contains several cellular subpopulations, including AT-MSCs, endothelial cells, and hematopoietic cells which represent a large portion of the fraction (20-50%).¹²⁸

Several clinical studies using SVF or only AT-MSC have been produced in the osteoarticular, cardiovascular, immunomodulation and wound healing field. Most successful results are currently in the area of modulating immune system and inflammation: treatment of autoimmune encephalomyelitis ¹²⁹, arthritis ¹³⁰, perianal fistula ¹³¹ or host-versus-graft disease.¹³²

2.3.3.2 ROS influences AT-MSC differentiation (Paper 4)

Pre-adipocytes are differentiated from AT-MSCs. However, factors that regulate their proliferation and differentiation are not very well known. Factors as FGF2¹³³, TGFβ¹³⁴ and miARN ¹³⁵ have been demonstrated to play a key role. Through a literature review, we demonstrated that reactive oxygen species (ROS), an oxygen derived molecule, influence tightly MSCs differentiation: it increases adipogenesis while MSCs osteogenesis is blunted by ROS.¹³⁶ (Annexe: Paper 4) In the future, ROS modulators could be used to bring under control MSC differentiation for tissue engineering use or cell therapy.

2.3.3.3 PRP as an ideal culture media for AT-MSC expansion (Paper 5)

The possibility to obtain the SVF quickly and simply from adipose tissue on patient’s bedside presents a considerable benefit. Another advantage comes from the synergistic effects existing between different cellular populations in SVF. These advantages are offset, by the heterogeneity of this fraction since the cellular composition varies considerably depending on the patient, the difficulty to establish quality controls due to brief time allocated and the limited number of cells. Only cell culture would permit to have a more homogenous product and more cell numbers with a strong quality control. Moreover, although adipose tissue is highly concentrated on AT-MSCS, for most tissue engineering or cell therapy uses, the number of MSCs is not enough if collected directly from adipose tissue.

Ex vivo cell culture is therefore mandatory for most clinical applications of AT-MSCs. Cell expansion requires a basal medium supplemented with proteins, growth factors, and enzymes.

Classical protocols use culture media supplemented with xenogeneic additives (e.g., fetal calf serum or fetal bovine serum (FBS)). These non-autologous products present a potential risk of infection and immunological reaction. Furthermore, they are inefficient as MSCs culture is slow.

Therefore, a safe and effective culture supplement is urgently needed to comply at best with national and international regulatory agencies’ requirements for clinical applications of MSCs.

The ideal cell culture media could be an autologous product.

To define an autologous system for AT-MSC proliferation, we proposed the use of autologous PRP, a plasma riche on platelets known to secrete orchestrated growth factors. (Sect. 1.3.2) In a study, we assessed in vitro the efficiency of autologous PRP on AT-MSC proliferation in

comparison to the classical FBS-supplemented medium. We demonstrated that the culture media supplemented with PRP showed dose-dependent higher AT-MSC proliferation than did FBS. Twenty percent of PRP was the most effective concentration to promote cell proliferation.

This condition increased 13.9 times AT-MSC number in comparison to culture with FBS, without changing the AT-MSC phenotype, differentiation capacity, and chromosome status. We concluded that autologous PRP is a safe, efficient, and cost-effective supplement for AT-MSC expansion, and it should substitute current non-autologous culture media, even for other cells than AT-MSC.¹³⁷ (Annexe: Paper 5)

Then after we aimed to assess the mechanism of action of PRP on AT-MCSs proliferation and elucidate the extra- and intra-cellular pathways involved in that. We submitted recently a paper, in which we concluded that PRP regulates AT-MSCs proliferation mainly through secreted growth factors (PDGF-AB, FGF, TGFβ1, VEGF and MIF). And after binding to their specific receptors, they mainly activate AKT and Smad2 signaling pathways.

Figure 13 Schema of 3 fat grafting zones where PRP could improve fat grafting survival by increasing peripheral and central zone. (Courtesy of Lee L.Q.Pu)

3 Perspectives