Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation

Adipose-derived stem/stromal cells (ADSCs) were first characterised in 2001, and have since been widely studied and used as a major source of cells with regenerative potential, with characteristics similar to that of mesenchymal stem/stromal cells (MSCs) [1–4]. ADSCs are isolated as part of the aqueous fraction derived from enzymatic digestion of lipoaspirate (the product of liposuction). This aqueous fraction, a combination of ADSCs, endothelial precursor cells (EPCs), endothelial cells (ECs), macrophages, smooth muscle cells, lymphocytes, pericytes, and pre-adipocytes among others, is what is known as the stromal vascular fraction (SVF).

ADSCs, like MSCs, have shown promise in regenerative and reconstructive medicine [5–8]. Recent advances in the area of tissue regeneration have put SVF on a par and at times even above ADSCs [9–17]. For instance, in a study of erectile function in a rat model of cavernous nerve injury, SVF treatment showed superior statistically significant results compared to ADSC treatment alone, especially in smooth muscle/collagen ratio and in endothelial cell content [12]. The advantage of SVF over ADSCs is believed to be in two fundamental areas. Firstly, although similar in properties such as immunomodulation, anti-inflammatory, angiogenesis, and so forth, the distinctive, heterogeneous cellular composition of SVF may be responsible for the better therapeutic outcome observed in comparative animal studies [9–12]. Secondly, unlike ADSCs, SVF is much more easily acquired, without the need for any cell separation or culturing conditions. Thus, the therapeutic cellular product is instantaneously obtained and has minimal contact with reagents making it comparatively safer and subject to the fulfilment of lesser regulatory criteria. It should be noted that, whereas ADSCs find utility in both allogeneic and autologous treatments, SVF, owing to the presence of various cell types known to cause immunological rejection, is suitable for autologous treatments only.

Although almost all ADSCs are derived from the white adipose tissue (WAT), as covered in this review, the identification of progenitor cells in brown adipose tissue (BAT) of adult humans is fascinating and worth a mention [18, 19]. Termed as BADSCs (brown adipose-derived stem cells), these have been isolated from BAT deposits present in relatively inaccessible regions such as the mediastinum, and are capable of differentiating to metabolically active BA cells with differences in surface antigen expression as compared to WAT-originating ADSCs [18]. Current understanding of WAT and BAT define these cells with distinct functionalities, and thus translational avenues for ADSCs from either source should be compared to identify specific therapeutic targets and potential advantage of one over the other. Understanding of the molecular mechanisms behind either cell fate and the possibility of inter-conversion are interesting avenues of research with basic and translational implications [20, 21].

Despite the potential of SVF in regenerative medicine there are challenges to overcome. First is isolation of SVF, which needs a specialised infrastructure such as a clean room facility, equipment, reagents, and technical capabilities. These conditions limit the reach of SVF to only major hospitals in tier 1/2 cities, especially in a country such as India. In this regard, the up and coming point-of-care biomedical devices which can take lipoaspirate as their input and produce sterile, injectable SVF as output will be beneficial. Secondly, the method of isolating SVF is a vital roadblock in the approved use of SVF for therapeutic applications. Digestion of lipoaspirate is achieved by collagenase, and the presence of collagenase in the injectable product does not bode well with regulatory authorities such as the US Food and Drug Administration (FDA) [3]. Consequently, alternative methods are being explored with some encouraging outcomes [22–25]. Finally, characterisation of the regenerative cells of SVF has not reached a wide consensus. Organisations such as the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society of Cellular Therapy (ISCT) have been updating the surface antigen-based definition of SVF cells, where CD34 antigen, primarily associated with haematopoietic stem cells (HSCs), became an important marker of regenerative, MSC-like cells of the SVF [1, 26, 27].

In this review, using the broader topics of isolation and characterisation of SVF, we will touch upon some of the challenges and innovations in the field and comment upon the future of SVF.

Criteria for characterising the cellular contents of SVF using surface antigen (cluster of differentiation (CD)) combinations is an evolving area of research as, within certain generally accepted norms, it differs between laboratories. A list of commonly used positive and negative markers identifying different cellular populations of SVF is provided in Table 1 [1, 26, 29]. Considering the variables present in isolation of SVF, such as the age of the patient, downstream processing, and so forth, the diversity observed between samples is quite understandable. However, if there is a relationship between the different ratios of cellular components present in SVF with its efficacy towards specific ailments, one might be able to come up with an optimum composition corresponding to the highest therapeutic efficacy. Traktuev et al. demonstrated that certain factors produced by ADSCs such as vascular endothelial growth factor (VEGF) help in migration, and that better survival of EPCs and correspondingly platelet-derived growth factors (PDGF)-BB produced by EPCs enable ADSCs to proliferate and migrate [46, 47]. They also provide proof of physical interaction between ADSCs and ECs in which ECs form a stable tubular, vasculature-like structure with support from ADSCs, both in vitro and in vivo [47]. This information along with some other articles has been used to draw up a schematic in Fig. 1 for the action of SVF, focussing on the interaction between ADSCs and EPCs [46–49].

ADSCs in SVF are currently defined to be positive for classical MSC markers such as CD73 and CD90, and express CD34 but not the pan-haematopoietic lineage marker CD45. CD34 is expressed by progenitors of haematopoietic and endothelial lineages as well, and in ADSCs it is expressed transiently up to about 8–12 population doublings in culture [1].The case of CD34 is interesting since it is still largely considered to be a marker for HSCs owing to its historical association with the enrichment of such cells for bone marrow and umbilical cord blood transplantation. Even the pericytic theory related to MSCs and ADSCs has two sides [50]; whereas Crisen et al. attribute CD34^– pericytes to be the progenitors of such stromal cells [51], Traktuev et al. demonstrated a CD34⁺ pericytic identity for ADSCs [46]. Maumus et al. tried to investigate this further but found that native CD34⁺ ADSCs did not exhibit in vivo pericytic markers, but they were rather observed over the course of the culture process [52]. Our data also show that both manually isolated and Stempeutron™-isolated SVF contains a CD146⁺ pericytic population that are mostly (>90%) CD34^– [29], suggesting that freshly isolated SVF contains a pericytic population devoid of expressing both CD34 and CD31 markers. Whether the CD146⁺ cells observed within the SVF population subsequently become CD34⁺ ADSCs remains to be determined. Considerable evidence also exists in favour of CD34 expression in bone marrow-derived MSCs (BMMSCs), especially in the early stages of BMMSC research which included data on the disappearance of CD34 upon culturing [53]. Many aspects of this puzzle are yet to be solved, but it is probable that CD34 marks different progenitor cell types such as different MSCs and vascular endothelial progenitor cells.

The first clinical applications of SVF were reported around 2007 to 2008 for cosmetic breast augmentation and also in the treatment of radiation injury post-radiotherapy in breast cancer patients [14, 65]. The Yoshimura group coined the term CAL, or cell-assisted lipotransfer, in 2008, where they enhanced fat grafts with SVF, demonstrating improved graft retention [14, 17]. Since these two early clinical reports from the last decade, there has been a many-fold increase in basic research and, consequently, many clinical trials are also now underway.

A superficial glance at the treatments highlights the two most preferred pathways, that is employing the vasculogenic and the immunomodulatory properties. We are yet to fully explore the multipotent properties of SVF cells which will only increase the breadth of their application. One recent example of enhanced osteoinduction by using SVF for dental implant surgery in human subjects provides encouraging results, wherein researchers found bone formation on implanting artificial graft material with SVF supplement compared to the graft alone [66]. The use of matrices/scaffolds and populating those with SVF and/or ADSCs is a promising area of application, though still in experimental phases [13, 78, 79]. Here, we will not go into much detail regarding the applications as that has been well accomplished in a recent two-part review [3, 26].

Clinics all across the globe began providing “fat stem cell”-based therapy shortly after its discovery, promising miraculous results and more, but often running into controversies [80–86]. Such therapies in the US are known to charge anywhere from USD5000 to USD100,000, and, although mostly harmless and sometimes beneficial, there have been reports of vision loss, tumours, and even deaths [80–86]. Being a major issue in the USA, the FDA had to step in with a draft guideline late in 2014 [87]. These guidelines can be considered in future development of technologies and procedures related to SVF and therapies. Although the “stem cell therapy” genre includes many types of stem cells, ADSCs remain the most marketed variety in the US [88].

The common practices of enzymatic and mechanical disruption of adipose tissue for isolating SVF are explicitly mentioned in the FDA document as “more than minimal manipulation” [87]. As and when the guidelines are implemented, SVF isolated by current protocols (enzymatic digestion) can be treated as a Category 351 product, that is a “drug/biologic” and in need of complete FDA regulation [68]. This calls for exploration of alternate methods, keeping in mind that regulations in the US often trickle down to other geographies, especially in matters of food and drugs.

Introduction of the Reliable and Effective Growth for Regenerative Health Options that Improve Wellness (REGROW) Act [89] in the US Senate last year led to scientific and policy debate, with prominent organisations such as the ISCT, the International Society for Stem Cell Research (ISSCR), and many patient and advocacy groups refusing to support it, at least in its current form [88, 90–94]. The REGROW Act aims to hasten the “conditional approval” of certain cell and tissue therapeutic products which demonstrate “reasonable expectation of effectiveness” along with a few other criteria [89]. However, the use of open-ended terms such as “reasonable expectation of effectiveness” amounts to a lack of clear scientific definition, thus leaving scope for interpretation of the law, consequently leading to potential abuse; such concerns are possibly behind this strong opposition towards the act.

Nevertheless, an urgent consensus is required among all stakeholders with regards to realising the translational potential of stem cells and other cell-based therapeutics, especially when it comes to serious unmet medical needs.

MSCs have been long known for their remarkable properties when it comes to regeneration and therapeutic potential. ADSCs are possibly the easiest to isolate among all the different types of MSCs in an adult human and in relative abundance too; up to 500 times more stem/stromal cells per gram as compared to a bone marrow source [95]. Simply put, ADSCs are potentially the most abundant regenerative cells in the human body and SVF is a step in the protocol to isolate ADSCs. As has been repeatedly mentioned in this review, the potential for use of both SVF and ADSCs in regenerative medicine are immense. However, care must be taken to go about it without harming the intended beneficiary, that is the patients and public in general. Guidelines, such as the ones from US FDA and their counterparts elsewhere will be important parameters in judging new therapies and technologies being developed, and we ought to keep abreast of such issues. Technology development is the single most important factor to realise the full potential of any new therapy, and SVF-based therapy is no exception. At the same time, it is evident that we need a better understanding of SVF and ADSC biology. This is a continuous endeavour and will only help to better establish the core principles and mechanisms of SVF- and ADSC-based therapies. In the process, we are likely to discover newer applications apart from the plethora already identified. Combining these therapies with other technologies such as decellularised or three-dimensional printed scaffolds with the aim of transplantation will jump-start other areas of clinical and commercial developments.