Adipose tissue: stem cells and beyond.
This article highlights potential uses for harvested fat and describes the current state of the art regarding adipose stem cells.
Adipose Tissue↗
Biomedical subjects
Sunil S Tholpady
Publications and source records attributed to Sunil S Tholpady.
The cellular plasticity of human adipocytes.
Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.
Adipocytes↗
Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.
Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.
ATP Binding Cassette Transporter, Subfamily G, Mem↗
Aberrant bony vasculature associated with activating fibroblast growth factor receptor mutations accompanying Crouzon syndrome.
Fibroblast growth factor receptor mutations are associated with and, in fact, cause most syndromes presenting with craniosynostosis. This knowledge has resulted in a shift in the paradigm of suture fusion causation; it was thought previously that abnormal tensional forces arising in the cranial base caused fusion of the vault sutures, but it is now understood that aberrant intercellular signaling in the developing skull leads to abnormal suture morphogenesis. Although the mutations associated with these syndromes are known and the phenotypic consequences are well documented, the pathway from mutation to phenotype has yet to be elucidated. Surgical reconstruction is the primary treatment of craniofacial abnormalities associated with craniosynostotic syndromes such as Crouzon syndrome. In many cases, calvarial vault reshaping is dependent on the quality of the autologous bone available; however, the bone of patients with craniosynostosis syndrome is often more brittle, thinner, and less robust than cranial bone from nonaffected donors. The relation between syndromic craniosynostoses and this bone has not been previously described. In this study, the osteon and blood vessel diameters of calvarial bone from patients with Crouzon syndrome and age- and sex-matched normal calvarial bone are measured. Statistical analysis demonstrates a quantitative and significant difference in the blood vessel diameter but not in the osteon diameter. This finding could be a result of abnormal blood vessel development caused by the fibroblast growth factor receptor mutation occurring before and coincident with bone formation and leading to weakened and fragile bone tissue.
Blood Vessels↗
BMP-9-transduced prefabricated muscular flaps for the treatment of bony defects.
Autologous bone grafting techniques involve the use of tissues that need to be extracted from healthy sites. This can lead to significant donor site morbidity that causes a one-site defect to become a two-site defect. Bone grafts can be especially difficult to manipulate, because bone is a relatively nonmoldable tissue. Furthermore, the inability of a bone graft to contain a transplantable vascular supply also limits the possible size that such a bone graft can be. Because of these limitations, a graft that was moldable with a vascular supply would possess significant advantages in reconstructive applications. In this research, gene therapy techniques were used to create such a graft. An adenovirus expressing BMP-9 was injected into the latissimus dorsi of a nude animal to cause bony differentiation of that muscle. Differentiation of the muscle to cartilage in bone was measured by reverse transcription polymerase chain reaction and immunohistochemistry to determine the optimal time of flap elevation. After injection of the BMP-9 virus, the animals were biopsied weekly over a 3-week period. Both bone and cartilage markers were discovered in these tissues over the study period. Optimal flap elevation time was established to be 2 weeks after injection of the virus.
Animals↗
Ex vivo fat graft preservation: effects and implications of cryopreservation.
There are a variety of recommended methods for harvesting, treating, and utilizing autologous fat grafts. Previous work with the MTT assay illustrated that various preimplantation handling techniques had minimal effect on the viability of fat samples. This assay was used to test the viability of harvested fat samples after being stored for up to 8 days in a variety of conditions. Surprisingly, freezing the fat before assaying also had no measurable detrimental effect, which led us to study this phenomenon in greater detail. The results demonstrated that fat stored at subzero temperatures showed remarkable maintenance of their mitochondrial metabolic activity as compared with fat stored in a 32 degrees C incubator. These data suggest exciting possibilities for storage and banking of human adipose tissue, which would reduce patient cost, discomfort, and time associated with multiple grafting procedures.
Adipose Tissue↗
Regulation of cranial suture morphogenesis.
The cranial sutures are the primary sites of bone formation during skull growth. Morphogenesis and phenotypic maintenance of the cranial sutures are regulated by tissue interactions, especially those with the underlying dura mater. Removal of the dura mater in fetuses causes abnormal suture development and premature suture obliteration. The dura mater interacts with overlying tissues of the cranial vault by providing: (1) intercellular signals, (2) mechanical signals and (3) cells, which undergo transformation and migrate to the suture. The intercellular signaling governing suture development employs the fibroblast growth factors (FGFs). In rats during formation of the sutures in the fetus, FGF-1 is localized mainly in the dura mater, while other FGFs are expressed in the overlying tissues. By birth, FGF-2 largely replaces FGF-1 in the dura mater. FGFs present in the calvaria bind either the IIIb or IIIc mRNA splice variants of the FGF receptors (FGFRs) 1, 2, or 3. Monoclonal antibodies to the b variant of FGFR2 were used to determine the distribution of FGFR2IIIb during suture development and its extracellular localization. FGFR2IIIb is present in association with mature osteoblasts and osteogenic precursor cells of the suture in the fetus. Ectodomains of FGFR2IIIb, the products of proteolytic cleavage of the receptors, were present throughout the extracellular matrix of sutures resisting obliteration (coronal and sagittal), but absent from the core of sutures undergoing normal fusion (posterior intrafrontal). This observation is consistent with a possible mechanism, in which truncated receptors bind FGFs, thus regulating free FGF available to nearby cells. Mechanical signaling in the calvaria results from tensional forces in the dura mater generated during rapid expansion of the neurocranium. Posterior intrafrontal sutures of rats, which fuse between days 16 and 24, were subjected to cyclical tensional forces in vitro. Significant delay in the timing of suture fusion and increases in the expression domains of FGFR1 and 2 were observed, demonstrating the sensitivity of suture patency to mechanical signals and a possible role of the FGF system in mediating such stimuli. Finally, cells of the dura mater beneath the intrafrontal and sagittal sutures were observed to undergo a morphological transformation to a dendritic morphology and migrate into the suture mesenchyme between days 10 and 16 of development. This process may participate in suture and bone morphogenesis and influence the patency of the sutures along the anterior-posterior axis.
Animals↗