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Variation in Osteogenic Differentiation Capacities of Adipose-derived Stromal Cells by Anatomic Depot

Dalton Pham, Alan Nguyen, Jia Shen, Vi Nguyen, Michelle A. Scott, Aaron W. James

Dalton Pham, Alan Nguyen, Vi Nguyen, Aaron W. James, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, the United States
Alan Nguyen, Jia Shen, School of Dentistry, University of California, Los Angeles, California 90095, the United States
Michelle A. Scott, Nationwide Children’s Hospital, Columbus, Ohio, the United States

Correspondence to: Aaron W. James, MD, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Ave., 13-145 CHS, Los Angeles, California 90095, the United States.
Email: Awjames@mednet.ucla.edu
Telephone: +310-206-6754
Received: February 16, 2016
Revised: April 15, 2016
Accepted: April 17, 2016
Published online: June 23, 2016

ABSTRACT

Although bone marrow has long been studied as the primary source of mesenchymal stem cells (MSC), adipose tissue has been increasingly studied as a rich source of tissue resident MSC. Since the first description of adipose-derived MSC, or adipose-derived stromal cells (ASC), it has been clear that ASC have significant advantages for clinical translation, including increased cell frequency, high growth potential, and residence in a dispensable and accessible location (subcutaneous fat). Nevertheless, it is increasingly clear that ASC differ by anatomic location, although the exact differences between anatomic depot are not entirely agreed upon. Striking differences exist between subcutaneous and visceral fat depots, but even more interesting between various anatomic regions within subcutaneous fat. Understanding the specific manners in which ASC differ by location is especially important in the context of tissue engineering. In summary, the current literature regarding ASC clearly shows that not all fat is created equally. Clear cut, and for the most part reproducible, differences exist in the osteogenic potential, adipogenic potential and proliferation of ASC depending on their site of origin. How this information should be used is still a matter of uncertainty.

Key words: Adipose-derived stromal cells; Anatomic depot

© 2016 The Authors. Published by ACT Publishing Group Ltd.

Pham D, Nguyen A, Shen J, Nguyen V, Scott MA, James AW. Variation in Osteogenic Differentiation Capacities of Adipose-derived Stromal Cells by Anatomic Depot. International Journal of Orthopaedics 2016; 3(3): 549-556 Available from: URL: http://www.ghrnet.org/index.php/ijo/article/view/1608

Introduction

Although bone marrow has long been studied as the primary source of mesenchymal stem cells (MSC), these bone marrow-derived stem cells (BMSC) present a number of drawbacks. Autologous tissues such as BMSC are often limited in availability, and require operation with a risk of morbidity[1,2]. By contrast, adipose-derived stromal cells can be isolated from the stromal vascular fraction of lipoaspirate, and has been increasingly studied as a rich source of tissue resident MSC. In 2001, Zuk et al described this population of culture-derived, multipotential MSC from subcutaneous human white adipose tissue, termed adipose-derived stromal cells (or ASC)[3]. Since this time, ASC have been of great interest to investigators due to their relative abundance and accessibility being derived from adiposetissue, in contrast to BMSC[3-7]. Unlike BMSC, ASC are also advantageous in that they present minimal morbidity[1]. Additionally, ASC proliferate easily, expand quickly, and possess stable multipotency through multiple passages[3,8-10]. To date, ASC have been studied extensively in the context of bone tissue engineering,with the literature showing various strategies that have been implemented to better induce osteogenic differentiation in adipose tissue, including the introduction of growth factors, different substrates, and even genetic modification. For example, ASCcan easily be directed towards osteogenic differentiation via growth factor or substrate treatment during cell culture with ascorbic acid, β-glycerophosphate, bone morphogenetic protein-2, and/or other osteoinductive factors to name a few[11-22]. Furthermore, there are a numerous strategies that can be employed to achieve genetic manipulation for the purposes of upregulating osteogenesis; specifically, the vector and the gene delivery product can be selected[23]. For example, among different vectors available, viral delivery is a well-studied platform for gene transfer, while non-integrating methods have also been explored as it addresses safety concerns from the former[24,25]. Furthermore, in terms of products for viral delivery, these can range from micro (mi)RNAs to bone morphogenetic protein (BMPs), some of which are potent inducers of osteogenesis[26,27]. Taken together, these studies validate the versatility and promising therapeutic potential of ASC in a number of applications.

Adipose tissue is distributed throughout the body and can be categorized into the subcutaneous and visceral adipose depots. The visceral depot can be further subdivided into omental, mesenteric, retroperitoneal, intrathoracic, and gonadal adipose depots, among others[28]. As a result of the numerous studies regarding osteogenic properties of ASC, findings have increasingly pointed to the idea that adipose tissue harvested from different adipose depots possess variable osteogenic capacities, which may be due to the inherent characteristics that differ based on anatomical locations such as vascularization and cell population heterogeneity[29-33]. While it would be most ideal to use a well-identified and homogenous population for regenerative therapy, the ASC population remains quite heterogeneous[34]. In fact, one of the major limiting factors in its clinical application, which includes its osteogenic potential for bone regeneration, is its lack of clear parameters to define this stem cell population[35]. One of the gold standard approaches currently utilized is fluorescent-activated cell sorting (FACS), which uses surface markers to select for CD34+ and CD31- for ASCin general[35]. This however can present complications since surface markers may change from passage to passage, thus lowering overall yield[36]. The marker profile may further change with differences in the depot of origin. For example, intrathoracic, subcutaneous, and omental ASC are strongly positive for CD44, CD73, CD90, and CD29 markers with low CD31 expression[32,36-38]. Subcutaneous and omental ASC also consistently express high levels of CD105[39,40]. However, ASC isolated from pericardial intrathoracic depots had a significantly higher proportion of CD34+ cells compared to both subcutaneous and omental ASC[32,40] (Please see Table 1 for a summary on cell surface markers). Thus, comparative studies have begun to investigate the differences in ASC osteogenic potential both within subcutaneous depots from varying locations as well as across adipose depots. The objective of this review aims to assess the current literature with a focus on differences in osteogenic differentiation capacities of ASC derived from a variety of adipose depots, among other differences including cellularity, gene expression, and cell systems[41].

SUBCUTANEOUS ADIPOSE DEPOTS

A majority of the studies investigating the osteogenic potential of ASC have utilized subcutaneous adipose tissue as an ASC source due to its relative abundance and accessibility[29,32,33,42-52]. Within the subcutaneous depot, a wide variety of ASC harvest sites have been assessed, including abdominal[29,32,33,42,45,47,48,51], inguinal[49,50,52], thigh[51], dorsocervical[49], flank[51], gluteal[47], arm[51], and interscapular[46,52] regions. Even among subcutaneous adipose depots, there exists a number of differences including rates of proliferation and adipogenesis among other factors[41]. The emphasis of this review, however, is the osteogenic characteristics shared by these adipose depots. A study by Iwen et al comparing the osteogenic differentiation potential across subcutaneous adipose depots found that human gluteal subcutaneous (sc-)ASC exhibited higher alkaline phosphatase and proliferation rates than the donor-matched abdominal sc-ASC after 28 days[47]. Additionally, at this time point, mineralization was only present in gluteal sc-ASC, whereas it was absent in the abdominal sc-ASC[47]. Similarly, Levi et al found that abdominal sc-ASC had lesser osteogenic capacity compared to flank and thigh sc-ASC, with flank sc-ASC having the highest osteogenic potential, evidenced by alkaline phosphatase staining, bone nodule formation, and expression of a wide portfolio of osteogenic gene markers[51]. Another study comparing dorsocervicalsc-ASC and inguinal sc-ASC discovered that inguinal sc-ASC demonstrated superior expression of alkaline phosphatase and osteocalcin[49]. In another bone regeneration study utilizingsc-ASC in a large animal model (canine), autologous-derived sc-ASC were expanded ex vivo and seeded onto a coral scaffold; the cranial bone defect was repaired with growth of typical bone tissue in the canine model used[53]. Another study tested the efficacy of undifferentiated sc-ASC, as most other studies use ASC differentiated in vitro; applied to a scaffold in athymic nude rat calvaria, it was shown that undifferentiated ASC could still differentiate into osteocytes and osteoblasts given a scaffolding material[54]. Overall, although differences in each study exist, sc-ASC taken from the lower extremity (inclusive of gluteus, flank, thigh, and inguinal region) seem to demonstrate enhanced in vitro osteogenic potential.

Compared to other adipose depots, including visceral[29,42], omental[29,32,48], and intrathoracic[32], sc-ASC have consistently displayed greater osteogenic potential. Studies evaluating gene expression across multiple adipose depots showed that sc-ASC express significantly higher levels of the early osteogenic markers Runx2 and collagen I[29,32,42,45,46,48]. Furthermore, greater mineralization, osteocalcin, and osteonectin were observed in sc-ASC as well[29]. Not only did sc-ASC exhibit higher expression of osteogenic markers, but they also proliferated and differentiated into osteoblasts faster than ASC from other depots[29,33,42]. The more homogeneous cell population of spindle-shaped morphologies consistent with ASC from subcutaneous regions is hypothesized to underlie this difference in osteogenic differentiation rate, with the more heterogeneous visceral ASC depots exhibiting longer differentiation times[29]. In contrast, one study found that sc-ASC showed significantly lower alkaline phosphatase activity, proliferation rates, and mineralization than visceral (v-)ASC in rabbits[33]. However, it is important to note that sc-ASC from the same anatomic region in rabbits, rats, and humans did not display similar osteogenic potentials; rabbit sc-ASC displayed the lowest osteogenic potential out of the three model systems[50]. Thus, significant differences across species may complicate ASC comparisons. Remarkably, one clinical study has demonstrated successful reconstruction of craniomaxillofacial defects in 10 of 13 cases using human sc-ASC[55]. While sc-ASCshow promise as a potential therapy for bone repair, further large-animal studies and pre-clinical trials are required before they can be reliably employed clinically for bone repair.

VISCERAL ADIPOSE DEPOTS

An alternative source of ASC that has been well studied with respect to osteogenic capacity includes visceral adipose depots. In arecent study conducted by Jung et al involving visceral abdominal human ASC, the visceral ASC (v-ASC) were observed to have a more heterogeneous morphological makeup[29]. In addition to the spindle-shaped cells consistent with ASC, these cell cultures also contained contaminating cells, especially fibroblasts and mesothelial cells. These other cell types, however, were found to be transient in v-ASC cultures, decaying after only four passages. As a result of the heterogeneity of v-ASC, osteogenic differentiation was observed to be markedly lower than for abdominal human sc-ASC[29].

Studies have indicated that the specific tissue source indeed plays a role in determining the characteristics for that extracted cell population. Specifically, observed variations in proliferation, and regulation of adipogenic and osteogenic regulation are notable[42,56], and functional differences are hypothesized to be due to differences in adipose stem cell. For instance, in terms of adiposity and proliferation, the subcutaneous depot exhibited a significantly higher level than that for visceral depots, thus yielding better organized adipocytes[42]. The human sc-ASC showed increased secretion of adiponectin and were more resilient against lipolysis[42]. It is further hypothesized that such functional differences may be accounted for by metabolic differences observed between subcutaneous and visceral sources through a ‘memory’ of the fat pad of origin. Interestingly too, this difference does show significant correlation to variations in telomere length; in comparison to sc-ASC, v-ASC actually have been shown to express significantly longer telomere lengths which was strongly correlated to patient age. However, the differences in depot may be a reflection of the shorter telomere length of ASC cells[57]. Finally, one study observed a statistically significant difference in fibroblast colony-forming unit CFU-F ability between sc-ASC and v-ASC[58]; tissue harvested from subcutaneous sites had a yield of 2.3-fold more CFU-F/unit volume than those from visceral donor sites, thus indicating differences in stem and progenitor cell content[58]. Taken together, in contrast to sc-ASC, v-ASC exhibit lower levels of proliferation, adiposity, and osteogenic potential along with increased cellular heterogeneity, all of which may potentially be attributed to differences in ASC profile dependent on the site of origin. With a relatively high heterogeneity and markedly lower osteogenic capacity compared to sc-ASC, among other drawbacks, v-ASC do not appear to be as promising for clinical applications given other sources of ASC available.

OMENTAL ADIPOSE DEPOTS

The omental adipose depots are a subset of visceral adipose depots, which also include mesenteric, retroperitoneal, intrathoracic, and gonadal adipose depots[28]. Similar to the visceral abdominal human ASC cultures, the omental abdominal human ASC cultures in the study done by Jung et al also contained contaminating mesothelial cells at earlier time points in culture[29]. In fact, the majority of the cell cultures were mesothelial cells, with only a few spindle-shaped visceral-type ASC. The presence of the mesothelial cells in omental ASC cultures led to lower osteogenic differentiation, consistent with the idea that heterogeneous populations of different ratios of cell types may affect osteogenic differentiation potentials[29-31].The mesothelial cells proved to be short-lived, as evidenced by the predominantly visceral-type ASC in culture by the fourth and fifth passage; however, despite still exhibiting osteogenic differentiation potential, past studies have shown that osteogenic differentiation potential actually decreases as the number of cell passages increases[29,48].

In comparative studies against other sources of fat depots, ASC isolated from the omentum exhibit several notable characteristics. For example, in contrast to intrathoracic depots which show greater levels of adipogenic markers, ASC isolated from the omentum demonstrate greater osteogenic markers in culture[56]. Investigators believe that this difference is due to the inherent properties of such adipose cell progenitors. To account for differences of cell types from different depots, flow cytometry analysis was used to demonstrate that subcutaneous adipose tissue contained more adipose tissue-derived cells while the omentum contained increased blood derived cells[43]. Proliferation rates were noted to be similar from both depots, although the findings suggest an overall, lower differentiation capacity for the omental depot[43]. These findings have been confirmed via electrophysiological analysis testing for membrane potential and capacitance, again comparing donor-matched subcutaneous adipose tissue to donor-matched omental pads. In another study, a significant difference in proliferation and adipogenesis was found in the subcutaneous-derived population[59]. Interestingly, in regards to tissue ultrastructure, the omentum depot was found to be macroscopically less stiff and even more vascularized than those from the abdominal subcutaneous depot. Furthermore, omentum provided for a significantly higher stromal vascular fraction (SVF) yield[56], which may be explained by the presence of more endothelial cells in the vascularized omentum. Taken together, the differences in proliferation, adiposity and osteogenic potential betweenomentum derived ASC and other depots suggest that the origin of the depot has a profound effect on the predilection and characteristics of the ASC.

OTHER ADIPOSE DEPOTS

The buccal fat pad in the oral cavity, also known as Bichat’s fat pad, has also been identified as a viable source of ASC[60]. For example, Niada et al assessed the osteogenic potential of porcine ASC from the buccal fat pad compared to sc-ASC and found comparable increases in collagen production, calcified extracellular matrix, alkaline phosphatase, and osteonectin[46]. These findings are consistent with the findings of another study involving human ASC from the same region[45]. Nevertheless, the buccal fat pad presents the challenge of being limited in supply, along with difficulty of access being in proximity to important neurovasculature.

Aside from omental depots, other smaller depots of visceral adipose depots, such as retroperitoneal and intrathoracic, have not been studied as extensively. One study comparing sc-ASC to retroperitoneal perinephric ASC found that the retroperitoneal ASC had significantly lesser osteogenic capacity[49]. Similarly, regarding intrathoracic adipose depots, human pericardial ASC possessed superior osteogenic potential than thymic remnant ASC, though both were of inferior osteogenic differentiation capacity compared to omental and sc-ASC[32]. Interestingly, ASC from intrathoracic depots seemed to be oriented more towards adipogenic differentiation even in osteogenic conditions, as seen by spontaneous intracellular lipid accumulation, a phenomenon less commonly observed in the subcutaneous or omental ASC[32].

It is important to note that while retroperitoneal and intrathoracic adipose sources may function in theory, this does not hold practical clinical applications outside of basic biology. A feasible tissue engineering approach would not likely utilize such a source that is both difficult to access and limited in supply.

Discussion

The current literature regarding adipose tissue resident MSC (or ASC) clearly shows that not all fat is created equally. Clear cut, and for the most part reproducible, differences exist in the osteogenic potential, adipogenic potential and proliferation of ASC depending on their site of origin. How this information should be used is still a matter of uncertainty.

Firstly, differences between sc-ASC clearly exist between anatomic sites, with sc-ASC from the thigh/gluteus/flank appearing to be most primed to undergo osteogenesis. The reasons for this variation by anatomic site are still unclear. Differences in adiposity, vascularity, and relative isolation of superficial versus deep adipose tissue may play a role. Nevertheless, larger scale studies are needed to verify these findings. In the end, such information is clinically relevant, for example, in that a surgeon may have a better foundation for choosing materials for tissue engineering applications.

Even more striking are the differences in differentiation potential between sc-ASC and v-ASC. These comparisons are more meaningful from a basic biologic perspective, rather than a tissue engineering perspective. Certainly, sc-ASC are an obvious first choice for any tissue engineering application given their abundance and accessibility. Nevertheless, some reproducible differences do exist, including lower levels of proliferation, adiposity, and osteogenic potential along with increased cellular heterogeneity in v-ASC compared to sc-ASC. Furthermore, while studies exploring the effects of paracrine and endocrine signaling by ASC are few, one study did indicate that adipose tissue do secrete endocrine/paracrine activity to elicit effects on the skeletal regeneration[61].Specifically, the endocrine/paracrine activity of some adipose tissue consists of bone anabolic factors, which include insulin like growth factor binding protein (IGFBP)-2 and WNT10b[61]. Furthermore, between omental and preadipocytes, the secrotomes have been found to be distinct; the former has been found to induce more macrophage and monocyte chemoattraction through Interleukin 6 (IL-6) and Janus kinase (JAK)-mediated signaling[62]. Subcutaneous adipose tissue also had higher production of pro-inflammatory cytokines, including IL-6 and IL-8, in contrast to that of abdominal adipose tissue[63]. Another study evaluating the effect of origin site on angiogenic growth factor gene expression found that there was a specific correlation between adipocyte size, which was a function of depot location, andvascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), fibroblast growth factor-10 (FGF-10), and leptin mRNA levels[64]. In both subcutaneous and omental human adipose tissue derived, comparing obese patients versus non-obese patients, there was increased expression of tumor necrosis factor alpha receptor (TNFR)-60 in the former, which correlated with BMI and fat cell size[65-67]. Similarly, it has further been hypothesized that this balance between subcutaneous fat and visceral fat may influence systemic insulin resistance, likely through regulation of TNF[68]. Potential explanations for these differences have included the degree of vascularity, composition of ASC, presence of contaminating mesothelial cells, and endocrine differences with adiposity highly regulated by glucocorticoids[69,70].

While several studies have investigated the immunomodulatory capacity of ASC, particularly their role in increasing regulatory T cells[71], suppressing T cell function[72-75] and natural killer cell cytotoxicity[76], the exact sources of ASC used in these studies remain unspecified. While few studies exist elucidating the differences in immunomodulatory effects of origin-specific ASC, one study by Zhu et al recently demonstrated that ASC derived from omental and subcutaneous differ significantly in their inflammatory effects due to greater production of chemokines by omental ASC, which is responsible for inducing migration of macrophages and monocytes, likely through Interleukin 6 (IL-6) and Janus kinase (JAK)-mediated signaling[62]. The study also suggests ASC secretion of exosomes via identification of typically intracytoplasmic proteins in the culture medium[62]. This hypothesis would be consistent with prior findings of human ASC-derived exosomes’ immunomodulatory role via inhibition of T cell proliferation, differentiation, and activation[72]. Similarly, in another study by Cohen et al, various intra-abdominal fat depots were found to have distinct immunomodulatory micro­environments[77]. Specifically, the leukocyte and progenitor populations were compared, and omental adipose tissue had nearly two-fold more leukocytes and a significantly higher proportion of T-cells than its parametrial and retroperitoneal counterparts[77].

Furthermore, several limitations exist regarding the current literature in comparing ASC depots. For example, there is disagreement in which subset of omental depot is most osteogenic or adipogenic. Not only is there a lack of consensus in terms of clear trends, there is also the underlying problem of a lack of standardization in methods[32]. Specifically, extraction protocols, culture conditions, number of passages, and donor demographics are all variables to consider in comparing studies[32]. Furthermore, species of ASC derivation is a potential complication factor, as ASC do differ based on species. Several investigators have postulated that anatomical characteristics of each adipose depot such as heterogeneity of cell populations and vasculature may affect the corresponding ASC osteogenic differentiation potentials[33,43]. Some studies, on the other hand, have found conflicting results, where non-subcutaneous ASC exhibited either stronger or similar osteogenic indicators compared to ASC from subcutaneous adipose depots[32,33,44,46]. In fact, one study found that all ASC possessed the same osteogenic differentiation potential within an individual donor[78]. Culture methods too, along with cell isolation techniques and degree of cellular heterogeneity may differ between studies.Considering that the present studies reviewed have also used donor-matched ASC comparisons, this finding brings up an essential point of consideration in the complexity of assessing the osteogenic potential of ASC. Not only does depot-dependent variability exist for osteogenic capacity of ASC, but donor-dependent differences are also a large contributing factor as well. A myriad of donor-dependent factors have been documented as affecting osteogenic potential of ASC, including obesity/body mass index[79-82], osteoarthritis[81,83], and gender[84]. Interestingly, donor age did not seem to have a significant effect[85,86]. Additionally, the complexity of drawing comparisons between variousASC populations for consensus is further exacerbated by the absence of standardization in research methods and practices pertaining to ASC extraction, culture conditions, and especially the number of passages ASC undergo before being evaluated, which has been found to have a significant impact on osteogenic potential[48]. In aggregate, researchers must take into consideration this multitude of contributing factors while evaluating a wide array of osteogenic indicators to obtain a better representation of the efficacy of certain ASC in bone-forming applications.

In terms of clinical applications, there are some concerns regarding the safety of using ASC in regenerative therapy. For one, the literature has shown that excessive adiposity has been associated with increased incidence of breast cancer in post-menopausal women, likely due to IL-6 secretion by ASC which is hypothesized to promote migration and invasion of breast cancer cells[87,88]. Another study performed ex vivo detected chromosomal abnormalities after extended ASC expansion in culture[89], similar to the findings of other groups that culture propagation of MSC introduces genetic instability and tumorigenicity[90]. Furthermore, one ASC study pertaining to bone formation in canine by Kang et al found cytologic abnormalities along with increased proliferation associated with ASC[91,92]. With regards to muscle formation, human-derived ASC applied to a murine model demonstrated increased inflammation in cardiac muscle[93]. Lastly, in the context of cartilage repair, Jung et al demonstrated that human-derived ASC applied in a murine model did not trigger any changes in proliferation rate, inflammation, tumor formation, or cytologic abnormalities[94]. On the contrary, an increasing number of recent Phase I and IIb studies focusing on the safety of ASC clinical application do not identify any significant risks or adverse effects in the short term[95]. However, it is important to note that the standardization of laboratory practices for ASC is lacking; even for MSC, cell proliferation and local inflammation are assessed in less than one-third of the manuscripts, and of the safety assessment manuscripts, MSC comprise of 85% while ASC constitutes less than 15%[96]. Taken together, while some studies report that ASC in clinical applications have not shown any significant risks, others have shown effects on increased inflammation and cytologic abnormalities which would require further investigation.

In terms of future direction of ASC therapy, with its discovery nearly 16 years ago, research in ASC application has made significant progress to provide for a promising stem cell therapy tool[95] and interest continues to increase. Regenerative applications of ASC are not limited to bone; ASC have been applied to cartilage repair[97], cardiovascular regeneration[98], and even hair[99] among other tissue types. With such versatility to repair and regenerate various tissue types, therapies using ASC has offered a paradigm shift that may provide an alternative solution to numerous diseases. With their relative abundance and ease of harvest from adipose tissue, ASC when combined with other tissue engineering techniques such as scaffolds and growth factors provide great promise as a therapeutic tool for the future[100], and will hopefully overcome the existing limitations surrounding the conventional approaches used in reconstruction surgery.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

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Peer reviewers: Gaël Rochefort, PhD HDR, EA2496, Faculté de chirurgie dentaire, Université Paris Descartes, 1, rue Maurice Arnoux, 92120 Montrouge France; Rubén Daniel Arellano, PhD, Full time Professor Researcher Medicine School, Torre, México; Andrea Del Fattore, PhD, Regenerative Medicine Area, Children’s Hospital Bambino Gesù, Padiglione Giovanni Paolo II, Lab Ricerca, piazza Sant’Onofrio 4, 00165, Rome, Italy.

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