RESUMEN
We previously discovered some adipocytes in the major white fat depots of mice and humans arise from bone marrow-derived cells of hematopoietic lineage rather than conventional mesenchymal precursors, termed bone marrow-derived adipocytes (BMDA). Here we aimed to determine if hematopoietic lineage cells isolated from adipose tissue and circulation of humans could undergo adipogenic differentiation in vitro, thereby establishing an in vitro model for studies of BMDA. We hypothesized that hematopoietic lineage cells isolated from adipose tissue, but not circulation, of humans would demonstrate adipogenic potential. Participants included younger (20-50 years) and older (>50-75 years) men and women, BMI 20-37 kg/m2. Subcutaneous abdominal adipose tissue biopsies were obtained and stromal cell populations identified by flow cytometry. Sorted cells underwent in vitro cultivation via traditional mesenchymal culture methodology (mesenchymal lineage) or a novel 3D-fibrin clot followed by traditional adherent culture (hematopoietic lineage) for assessment of proliferation and differentiation capacity. We found hematopoietic lineage cells isolated from the adipose tissue stroma, but not the circulation, were capable of proliferation and multilineage (adipogenic and osteogenic) differentiation in vitro. We provide a new investigative tool that can be used to perform translational studies of BMDAs and provide initial evidence that hematopoietic lineage cells isolated from the adipose tissue of humans can undergo hematopoietic-to-mesenchymal transition with multilineage differentiation potential in an in vitro environment.
RESUMEN
White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor-derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte-restricted adiponectin gene promoter was adoptively transferred to wild-type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal-vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.
Asunto(s)
Adipocitos Blancos/fisiología , Tejido Adiposo/fisiología , Células Madre/fisiología , Animales , Células de la Médula Ósea/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Fusión Celular/métodos , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Células Madre Hematopoyéticas/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genéticaRESUMEN
Analysis and isolation of adipocytes via flow cytometry is particularly useful to study their biology. However, the adoption of this technology has often been hampered by the presence of stromal/vascular cells in adipocyte fractions prepared from collagenase-digested adipose tissue. Here, we describe a multistep staining method and gating strategy that effectively excludes stromal contaminants. Initially, we set a gate optimized to the size and internal complexity of adipocytes. Exclusion of cell aggregates is then performed based on fluorescence of a nuclear stain followed by positive selection to collect only those cell events containing lipid droplets. Lastly, negative selection of cells expressing stromal or vascular lineage markers removes any remaining stromal contaminants. These procedures are applicable to simple analysis of adipocytes and their subcellular constituents by flow cytometry as well as isolation of adipocytes by flow sorting.
Asunto(s)
Adipocitos/citología , Linaje de la Célula/genética , Separación Celular/métodos , Citometría de Flujo/métodos , Tejido Adiposo/citología , Biomarcadores , Diferenciación Celular/genética , HumanosRESUMEN
It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity.