Characterization of transplanted green fluorescent protein+ bone marrow cells into adipose tissue

Abstract

Following transplantation of green fluorescent protein (GFP)-labeled bone marrow (BM) into irradiated, wild-type Sprague-Dawley rats, propagated GFP(+) cells migrate to adipose tissue compartments. To determine the relationship between GFP(+) BM-derived cells and tissue-resident GFP(-) cells on the stem cell population of adipose tissue, we conducted detailed immunohistochemical analysis of chimeric whole fat compartments and subsequently isolated and characterized adipose-derived stem cells (ASCs) from GFP(+) BM chimeras. In immunohistochemistry, a large fraction of GFP(+) cells in adipose tissue were strongly positive for CD45 and smooth muscle actin and were evenly scattered around the adipocytes and blood vessels, whereas all CD45(+) cells within the blood vessels were GFP(+). A small fraction of GFP(+) cells with the mesenchymal marker CD90 also existed in the perivascular area. Flow cytometric and immunocytochemical analyses showed that cultured ASCs were CD45(-)/CD90(+)/CD29(+). There was a significant difference in both the cell number and phenotype of the GFP(+) ASCs in two different adipose compartments, the omental (abdominal) and the inguinal (subcutaneous) fat pads; a significantly higher number of GFP(-)/CD90(+) cells were isolated from the subcutaneous depot as compared with the abdominal depot. The in vitro adipogenic differentiation of the ASCs was achieved; however, all cells that had differentiated were GFP(-). Based on phenotypical analysis, GFP(+) cells in adipose tissue in this rat model appear to be of both hematopoietic and mesenchymal origin; however, infrequent isolation of GFP(+) ASCs and their lack of adipogenic differentiation suggest that the contribution of BM to ASC generation might be minor.

Figure 1. Evaluation of the distribution and ultrastructure of bone marrow-derived GFP⁺ cells within the fat of radiation chimera rat 165 days following bone marrow transplantation

Panel 1A. Tissue was stained for SMA (red), F-actin (blue) and the nucleus (Hoechst’s dye, cyan) in addition to collecting the endogenous fluorescence of GFP (green). Single slice confocal of the tissue shows GFP⁺ cells were evenly scattered around the 70–100 μm adipocytes (some labeled A as reference). Most GFP⁺ cells were observed to be SMA positive (arrows) as more clearly shown in the black and white rendition of the red channel in the Panel A insert. The SMA signal in the GFP+ cells was equal to that observed in the pericytes surrounding the blood vessels (arrowhead). Panel 1B. Partial confocal stack showing a GFP+ adipocyte in the BM chimera. This is a rare event and was observed only once in 6 chimeric animals examined (tissue counterstained for F-actin (rhodamine phalloidin, red) and nucleus (Hoechst’s dye, blue). GFP signal is cytoplasmic and does not partition into the lipidic inclusion of the adipocyte. Panel 1C. Expanded XY projection from Panel B at axes delineated by the horizonal and vertical lines, indicating the GFP⁺ blood cells within the F-actin⁺ blood vessels (arrows). The GFP⁺ cytoplasmic labeling of the positive adipocyte surrounds the lipidic portion of the cell (*). Panel 1D. Transmission electron micrograph of the typical GFP⁺ cell within the adipose tissue, showing these cells, integrated between adipocytes, possess large quantities of rough endoplasmic reticulum. Panel 1E. Immuno-TEM analysis of LRWhite acrylic embedded chimeric adipose tissue indicated that these cells were GFP⁺ when stained for the GFP protein (arrows indicate secondary antibody 5 nm gold particles).

Figure 2

Adipose tissue from GFP⁺ chimeric rats was stained for CD90 (Cy3, red) and counterstained F-actin (blue). Areas within the tissue show variation in colocalization of CD90 with the GFP BM marker. Left top panel, arrows indicate dual signal, arrowhead indicates GFP⁻/CD90⁺ cell. Lower bottom panel indicates black and white rendition of red channel. Right top panel shows an area without apparent colocalization of CD90 and GFP signal.

Figure 3

Adipose tissue from GFP⁺ chimeric rats was stained for CD45 (Cy5, blue) and counterstained F-actin (red). Left top panel shows single optical slice confocal image and right panel shows the black and white rendition of the Cy5 channel, accentuating the CD45+ cells. Arrows indicate the cells that are both GFP⁺/CD45⁺, arrowheads indicate the cells that are GFP⁺/CD45⁻. In this panel there are also a few GFP⁻/CD45⁺ cells in this field with this phenotype. The bottom panels are higher magnification of the top panels.

Figure 4

a) Light micrograph of isolated cells from adipose tissue at passage 3, b) corresponding image with GFP⁺, c) Light micrograph of adipose stem cells that had undergone adipogenesis, and d) corresponding image with GFP⁺.

Figure 5

Surface markers expression of cultured, passage three ASCs from naïve GFP⁺ rats. a) Representative plots of ASCs obtained by culturing the subcutaneous and abdominal fat tissues obtained from naïve animals. Flow cytometric analysis showed that cultured ASCs expressed CD29 and CD90. b) Cells expressed CD29 and CD90 (red) in vitro, while the majority of these cells were negative for CD31, CD45, CD106 and CD133. Blue is F-actin stain and green is endogenous GFP signal. Inserts are the black and white rendition of the red channel for each of these markers.

Figure 6

Surface markers expression of cultured, passage three ASCs from GFP⁺ radiation chimera. a) ASCs obtained from fat tissues of radiation chimera were analyzed by flow cytometry. CD29 and/or CD90 expressing ASCs were mostly GFP negative; however, small numbers of GFP positive bone marrow-derived ASC were detected. Of note, ASCs from the abdominal fat and subcutaneous fat differ in the expression of CD90. Data is mean ± SD of 3 experiments, * = p ≤ 0.05. b) Representative scattergrams showing GFP⁺ CD29⁺/CD90⁺ ASCs. The intensity of the expression of CD29 and CD90 on GFP⁺ ASCs is lower than that expressed on GFP⁻ ASCs. The abdominal and subcutaneous fats were obtained from radiation chimera at 161 days after bone marrow transplantation.