Mitochondria in White, Brown, and Beige Adipocytes
1University Children's Hospital Zurich, Tissue Biology Research Unit, August Forel Strasse 7, Zurich, Keywords Adipose-derived stem cells; Cell- based therapies; Skin Jo urn al o f Tissue Science &Eng in ee ring. ISSN: cells meet preadipocyte commitment: going back to the future. J Lipid Res. Feb;53(2) doi: /jlr.R Epub Dec 2. Adipose tissue stem cells meet preadipocyte commitment: going back to the. multipotent stem cells/progenitors named adipose tissue-derived stromal cells. Recent studies have .. A thin ring of cytoplasm. Mitochondria .. tissue stem cells meet preadipocyte commitment: going back to the future.
Heterogeneity of MSC isolations in general has been discussed in many publications [ 54 — 57 ]. Nevertheless, when analyzing the adherent population by flow cytometry, no macrophages, endothelial cells, lymphocytes, or granulocytes seem to remain [ 50 ]: However, endothelial cells in culture are extremely susceptible to culture conditions, such as supplements and particularly shear stress, and, therefore, may dedifferentiate or trigger apoptosis under static culture conditions [ 57 ].
The heterogeneity of cultured ASCs can be reduced by a washing procedure early in the beginning of the cell culture [ 58 ], indicating that several subsets require different time points to adhere to the cell culture plastic.
Other efforts to reduce the heterogeneity or to isolate specific subsets of ASCs were carried out by using flow cytometric sorting or immunomagnetic separation, either by positive or by negative selection [ 59 — 62 ]. The usage of such techniques for the reduction of heterogeneity is more or less beneficial but leads to a very small cell yield.
By using immunomagnetic beads, Rada and coworkers demonstrated that the SVF is composed of several subpopulations, which express different levels of ASC markers and exhibit varying osteogenic and chondrogenic differentiation potentials [ 60 ]. Cultured ASCs show an extensive proliferative ability in an uncommitted state while retaining their multilineage differentiation potential.
In later passages, ASC cultures are homogeneous and exhibit a fibroblastoid morphology. The composition of subpopulations, therefore, may change during expansion [ 63 ].
Cell culture selects for this homogeneous morphology, enriching for cells expressing a stromal immunophenotype [ 28 ]. Different studies have characterized and compared the immunophenotype of cultured ASCs in early and later passages over the past few years and found that the expression profile of ASCs changes during culture time.
It has been repeatedly shown that freshly isolated ASCs express different surface markers than ASCs in higher passages [ 2852 ].
At the beginning of the culture, ASCs do not uniformly express all surface proteins, which are supposed to be characteristic. Subsets with distinct phenotypic properties can be discerned in freshly isolated cells by specific surface markers [ 63 ].
The expression of the markers seems to be dependent on culture conditions or time in culture. The specific surface markers CD29, CD90, and CD increase during culture [ 28 ], while the expression of other markers decreases [ 2852 ]. It has also been described that only some ASCs lose their CD34 expression with increasing culture time and that cell culture in medium supplemented with acidic FGF maintained CD34 expression for at least 10—20 weeks [ 49 ].
On the other hand, expression of CD and especially CD is relatively low on the freshly isolated ASCs but rises to a high extent during cell culture [ 52 ].
Another study also described that stromal cell-associated markers CD29, CD73, CD are initially expressed lower but rise during successive passages, whereas the expression of CD34 dramatically decreases [ 28 ]. Whereas expression of some characteristic markers is consistently found to be expressed by cultured ASCs and others are consistently not found to be expressed summarized in [ 64 ]many studies differ in some of the markers.
The expression of some antigens is described in a very contrary way. These differing results are due to differences in the isolation or culture method or caused by the investigation of different passages of cultured ASCs: CD is described as lowly expressed by the whole ASCs population, and this expression decreases with culture time [ 28 ]. Since CD is also a marker for endothelial cells and pericytes, it could belong to a subset of ASCs [ 2649 ].
Taken together, comprehensive studies are needed to further characterize the whole expression profile of ASCs in different passages in detail. What about Standardization of the Isolation and Culture Procedure? Discrepancies in the results of studies from different laboratories may result from many different origins. First of all, ASCs are isolated from different donors.
These donors differ in age, body mass index, gender, ethnicity, and their medical history e. It has been shown, for example, that the body mass index correlates negatively to the number of stromal cells per gram and their differentiation capacity [ 66 ]. The liposuction procedure may differ between different clinics, the liposuction or biopsy side is different, and the time lapse until isolation procedure starts differs between the laboratories. It has been reported for ASCs that liposuction side, liposuction procedure, age, or body mass index play an important role in the cell yield, growth, and frequency of stem cells [ 2666 — 69 ], but it is not clear whether this favours different subsets in cultured ASCs.
All these variables may affect the composition of the isolated initial cell culture, but it is extremely difficult, if not impossible, to standardize these variables. On the other hand, the methods and quality of isolations of ASCs from different laboratories per se vary tremendously, resulting in a different composition of the initial cell culture. Finally, the culture procedure of isolated ASCs differs between the laboratories; at the present time, there is no unique and standardized culture protocol for the culture of ASCs.
There are many variables that impair the cultured cells or the composition of subpopulations in their undifferentiated state: Summary of cell culture parameters which affect the undifferentiated state of ASCs.
In vivo, many cell types are attached to soft materials, either other cells or extracellular matrices, but most of what is known about cell structure and function in vitro derives from studies of cells plated onto rigid substrates, such as plastic [ 70 ]. As a result, some aspects found in vitro are rarely if ever seen in vivo [ 70 ]. Differentiation of MSCs, for example, has been shown to be dependent on the substrate on which the cells are cultured.
Whereas MSCs on stiff substrates expressed markers of osteogenesis, MSCs on softer substrates expressed myogenic markers, and cells on the softest gels expressed neuronal markers [ 71 ].
Nevertheless, stiffness alone is not sufficient to fully differentiate cells. Furthermore, the influence of plastic coating with collagen or fibronectin has also been shown to influence the differentiation state of MSCs [ 72 ]. Therefore, more work about the optimal substrate and substrate stiffness to culture ASCs is highly desirable. Only limited information is available about which medium optimally expands ASCs by maintaining the undifferentiated stem cell character in vitro [ 73 — 75 ].
It has been shown in cultures of MSCs that basal medium, glucose concentration, quality of FCS, cell plating, and cell density highly affect the final outcome [ 76 ], resulting in the expansion of populations with totally different potential. These factors have also been shown to be expressed by ASCs in earlier studies [ 277778 ] and are related to the undifferentiated state of ASCs and also to pluripotency of stem cells in general. Nevertheless, a physiological glucose content is one variable which should be considered to be near to the in vivo situation.
Furthermore, a low calcium concentration and supplementation with antioxidants have been shown to accelerate the proliferation of ASCs, but it was not clearly shown that this culture medium did not alter the whole differentiation capacity of ASCs [ 79 ]. There are many concerns about the practicability of foetal calf or bovine serum infectious complications, host immune reactions related to a possible use of ASCs in human therapeutical approaches [ 84 ].
Additionally, human serum may be a source of pathogen contamination or immunoreactivity and shows batch-to-batch variability. Using defined cell culture medium is an urgent need in order to produce ASCs for clinical applications. The gold standard for culturing ASCs would be a medium absolutely free of animal serum or factors, with well-known ingredients. Parker and coworkers tested eight commercially available serum-free media developed for use with other cell lines for their ability to support growth of human ASCs [ 74 ].
None of the available media was sufficient for supporting cell growth as purchased, and none performed better as a base medium than their standard medium containing serum. Others described serial testing of new medium formulations containing human serum or platelet lysate or tested the use of animal serum- or xeno-free media for the culture of ASCs in regard to cell morphology, cell proliferation, phenotype, and differentiation potential [ 85 — 88 ].
As a result of these studies, there is obviously no favourable serum- and xeno-free medium for the expansion of ASCs retaining their undifferentiated state.
Rajala and coworkers, for example, described a xeno-free medium that induced significantly higher proliferation rates than medium containing allogeneic human serum [ 88 ].
This medium maintained the differentiation potential of ASCs.
Adipose tissue stem cells meet preadipocyte commitment: going back to the future
Nevertheless, the authors detected significant differences in the surface marker expression of ASCs cultured in xeno-free medium compared with human serum. Therefore, standardization of the isolation and culture procedure is highly needed for a good reproducibility of results from different laboratories and studies. ASCs can be cultured by serial passaging without losing their multipotent properties [ 27 ] and have the capacity to maintain chromosome stability in long-term cultures [ 89 ].
The transcriptional and molecular events triggering the mesodermal lineage-specific differentiation of stem cells are well known [ 98 — ]. ASCs have also been shown to be angiogenic and hematopoietic supporting cells [ — ].
The potential of ASCs to differentiate into lineages with nonmesodermal origin, although ASCs originate from the mesoderm, is even more exciting.
The differentiation potential of ASCs into cells of ecto- and endodermal origin has also been shown. Therefore, the term pluripotent stem cells would be more correct for ASCs rather than multipotentas a differentiation into cells from all three germ layers has been shown. Nevertheless, the morphology of ASCs is different to other pluripotent stem cells, and their ability to form teratoma has not been shown.
A variety of studies documented the induced in vitro differentiation into hepatocytes, pancreatic islet cells, neural cells, endothelial cells, and epithelial cells [ 165075— ]. Our studies have clearly verified that ASCs can enter the epithelial lineage when treated with retinoids [ 50 ], conditioned medium CM from renal tubular epithelial cells, or a mixture of growth factors [ — ].
In vivo differentiation of ASCs toward renal epithelial cells has also been shown in a renal ischaemia-reperfusion model [ ]. The multiorgan engraftment of transplanted ASCs has been shown, in combination with epithelial lineage differentiation [ ]. Advanced Search Abstract Phenotypic diversity may play an adaptive role by providing graded biological responses to fluctuations in environmental stimuli. We used single-cell imaging of the metabolizable fluorescent fatty acid analog 4,4-difluorobora-3a,4a-diaza-s-indacene BODIPY -C12 and fluorescent 2-[N- 7-nitrobenzoxa-1,3-diazolyl amino]deoxy-D-glucose 2-NBDG to explore cellular heterogeneity in nutrient uptake in white adipose tissue WAT explants of rhesus macaques.
Relative free fatty acid FFA transport activity correlated with the cellular levels of FFA transporter protein-1 and the scavenger receptor CD36 in individual adipocytes. In vitro incubation of WAT explants for 24 hours caused partial desynchronization of cellular responses, suggesting that adipocytes may slowly alter their differential nutrient uptake activity.
WAT from animals containing a homogeneous population of large adipocytes was nonmosaic, in that every adipocyte exhibited a similar level of BODIPY-C12 fluorescence, suggesting that the development of obesity is associated with the loss of heterogeneity in WAT.
- Stem Cells International
Triglyceride TAG synthesis in white adipose tissue WAT relies on the insulin-dependent uptake and esterification of free fatty acids FFAs and the uptake of glucose, the main substrate for the glyceride-glycerol backbone of TAG. The enlargement of adipocytes and cLD associated with obesity leads to the development of local WAT inflammation and insulin resistance, whereas weight loss associated with a reduction in adipocyte size improves insulin sensitivity 4 — 9.
WAT is compartmentalized into two major anatomical depots, ie, sc and visceral WAT, whose relative distribution and functional properties are affected by sex, age, and energy balance Classical brown adipocytes have the capacity to increase energy expenditure through uncoupling proteindriven uncoupling of oxidative metabolism from ATP production, known as thermogenesis, and possess unique morphological characteristics, including a multilocular appearance multiple LDs and high mitochondrial content.
The functional significance of this adipocyte type in adult humans is currently under intense investigation.
Adipose tissue stem cells meet preadipocyte commitment: going back to the future.
Although interdepot heterogeneity in WAT function is a well-established concept, differences in the behavior of individual adipocytes residing within the same cell population is not well understood, and studies addressing this have been limited to in vitro-differentiated adipocytes and rodent models.
For example, mouse 3T3-L1 adipocytes display a significant variability in adipogenic gene expression, insulin sensitivity, and LD size and number, suggesting the existence of different adipogenic states within the same population of cells 15 — Whether these differences in cellular states represent the natural variability in gene expression or artifacts of in vitro culture is unknown.
High and low lipid uptake phenotypes persisted after cell redifferentiation and correlated with distinct patterns of gene expression One of the best examples of in vivo intratissue heterogeneity is the fat-specific insulin receptor knockout mouse, whose WAT is polarized into small and large adipocytes, with each adipocyte subpopulation displaying a unique gene expression pattern. Interestingly, fat-specific insulin receptor knockout mice were protected against obesity and glucose intolerance 20 Heterogeneity in cellular responses may be caused by noise, resulting in stochastic and reversible changes in various cellular functions, or by genetic mutations or epigenetic programming in individual cells, resulting in stable cell subpopulations with distinct functional properties 22 — In fully differentiated nondividing cells, such as adipocytes, differences in function may arise from differentiation from distinct adipose precursors or from differences in the microenvironment that affect cellular function.