ability to ameliorate type II diabetes in lipodystrophic mice Adipocyte progenitors from different anatomical locations and embryological origins differ in their ability to ameliorate type II diabetes in lipodystrophic mice Dario R. Lemos1; Kevin Shen1; Ben Paylor1; Misha Hutton1; Fabio M. V. Rossi1,2 1The Biomedical Research Centre, University of British Columbia 2222 Health Sciences Mall, Vancouver BC, V6T 1Z3, Canada; 2Department of Medical Genetics, Faculty of Medicine, The University of British Columbia, 317 - 2194 Health Sciences Mall Vancouver, BC V6T 1Z3, Canada Introduction White adipose tissue (WAT) has been shown to be a rich source of both stem-cells and progenitor cells. Although it is generally accepted that fat derives from mesoderm, recent data suggesteded that adipocytes can develop from embryonic neural crest cells in culture (1). In the present study we performed lineage tracing using the WNT1-Cre/R26-YFP transgenic mouse (2) and found that a fat depot located in the cephalic region derives from the neural crest. In an attempt to identify potential adipogenic progenitors (AP) with a novel ontogeny, we studied the properties of a population of neural crest-derived progenitor cells (CD31- :CD45- :7 integrin- :CD34+:Sca1+) that are phenotypically identical to those previously identified in the (mesodermal) subcutaneous fat depot by our lab (3). In order to functionally compare these neural crest-derived APs with those from mesodermal origin, we transplanted purified progenitors of each type into lipodystrophic AZIP mice and monitored the evolution of the diabetic phenotype that characterizes this model. Materials and Methods Animals and Transplantation Procedure Wnt1-Cre mice were crossed to R26-YFP animals to obtain the Wnt1-Cre/R26-YFP mice. AZIP (F1) mice were obtained by crossing transgenic FVBN/J mice kindly provided by Dr Gavrilova to wild type C57BI/6 mice. In the transplantation experiments, 104 YFP+ or GFP+ double sorted APs from either Cp or Sc fat respectively were suspended in 25ml matrigel and transplanted by subcutaneous injection into the sub-scapular region of syngeneic, GFP negative mice (n=4 per group). Cell culture Cells were grown in high-glucose Dulbecco’s modified eagle medium (DMEM), supplemented with 2.5ng/ml bFGF (Invitrogen) and 20% FBS. Cells were seeded in tissue-culture treated plates coated with Collagen type 1 (Sigma). After sorting, cells were allowed to adhere for up to 3 days, after which the medium was changed. Media was changed every 2-4 days. For adipogenic differentiation, the medium was changed to mebFGF-free media supplemented with 0.25M dexamethasone, 0.5mM Isobutylmethylxanthine, 1g/ml insulin, 5M troglitazone and cultured for ten days. Antibodies The following monoclonal primary antibodies were used: anti-CD31 (clones MEC13.3 [BD] and 390 [Cedarlane Laboratories]), anti-CD34 (clone RAM34 [eBiosciences]), anti-CD45 (clone 30-F11 [BD]), anti-CD45.1 (clone A20 [BD]), anti-CD45.2 (clone 104 [eBiosciences]), anti-Sca1 (clone D7 [eBiosciences]) and anti-7integrin (clone CA5.5 [produced in-house]). Cells were stained with Hoechst 33342 (2.5mg/ml) and resuspended at ~1x107 cells/ml immediately prior to sorting or analysis. Figure 1. Identification of a neural crest-derived fat depot in the cephalic area of the adult mouse (A) Anatomical localization of the cephalic fat depot. (B) WNT1-driven YFP expression. (C) Expression of YFP was confirmed by immunofluorescence using a 2ry ab conjugated to Alexa Fluor 568. (D) WNT1-driven YFP expression in the adrenal gland medulla (autonomic nervous system). A B C D A B C Figure 3. Adipogenic differentiation of NC-derived progenitors. CD34+:Sca1+ progenitors were isolated by cell sorting and cultured in differentiation medium for 10 days (A) Detection of perilipin expression by immunofluorescence (B) Oil Red O uptake upon differentiation. (C) Expression of adipogenic markers was measured by Taqman real-time PCR. B A C A B Figure 2. Isolation of neural crest-derived progenitors from adult mice (A) Analysis of stromal vascular fractions from different fat depots by (A) fluorescence microscopy and FACS. (B) Prospective purification of a CD45-:CD31-:7-:CD34+:Sca1+ population of progenitors derived from the neural crest by FACS. Figure 4. Effect of neural crest- and mesoderm-derived adipogenic progenitors on type II diabetes in lipodystrophic mice. 5x104 YFP+ (Cp) or GFP+ (Sc) APs were isolated by double cell sorting and injected subcutaneously into AZIP mice. (A) Bright field images of subcutaneous engraftments (B) Neural crest-derived progenitors differentiate into mature adipocytes (C) Differential effect of newly generated adipocytes on serum insulin and glucose levels in AZIP mice. Conclusions A fat depot located in the cephalic region of the mouse is composed of adipocytes that derive from the neural crest. A population of neural crest-derived progenitors with adipogenic activity resides within the cephalic fat depot of adult mice, indicating that ontogenically related progenitors are specifically involved in the expansion/maintenance of this depot. Although NC-derived APs can generate mature adipocytes de novo, in vivo, they are less effective than mesoderm-derived APs in reverting lipodystrophy-induced diabetes. Intrinsic properties such as clonogenicity/adipogenicity, adipokine secretory activity, may account for these differences.