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Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots
Cytotherapy (2007) Vol. 9, No. 5, 459 467
Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots JK Fraser1, I Wulur1, Z Alfonso1, M Zhu1 and ES Wheeler2 1
Cytori Therapeutics Inc., San Diego, and 2Aesthetic Plastic Surgery Center, La Mesa, California, USA
Background Human adipose tissue has been shown to contain multipotent cells with properties similar to mesenchymal stromal cells. While there have been many studies of the biology of these cells, no study has yet evaluated issues associated with tissue harvest. Methods Adipose tissue was obtained from the subcutaneous space of the abdomen and hips of 10 donors using both syringe and pump-assisted liposuction. Tissue was digested with collagenase and then assayed for the presence of different stem and progenitor cell types using clonogenic culture assays, including fibroblast colony-forming unit (CFU-F) and alkaline phosphatase-positive colony-forming unit (CFU-AP). Paired analysis of samples obtained from the same individual was used to compare harvest method and site.
adipocyte-depleted fraction. There was considerable donor-to-donor variation in stem cell recovery. However, paired analysis of tissue obtained from different subcutaneous sites in the same donor showed that tissue harvested from the hip yielded 2.3-fold more CFU-F/unit volume and a 7-fold higher frequency of CFU-AP than that obtained from the abdomen. These differences were statistically significant. Discussion Harvest site influences the stem and progenitor cell content of subcutaneous adipose tissue. Keywords adipose adult stem cell, liposuction, subcutaneous depot.
Results Syringe suction provided significantly greater recovery of adipocytes and a non-significant trend towards improved recovery of cells in the
Introduction In recent years it has become evident that adipose tissue contains a population of stem and progenitor cells with differentiation capacity beyond adipogenesis [1]. For example, different groups have demonstrated the ability of adipose tissue-derived stem cells (ASC) to differentiate in vivo or in vitro into cells exhibiting features characteristic of osteoblasts, chondroblasts, skeletal myoblasts [2,3], cardiac myocytes [4], neuronal cells [5,6], hepatocytes [7], hematopoietic cells [8] and endothelial cells [9,10]. We and others have shown that at least some of these different lineages can be derived from single multipotent cells, indicating the presence of a novel stem cell population within adipose tissue [2,11].
Adipose tissue depots are generally separated into two categories, subcutaneous (or peripheral) adipose and visceral (or central) adipose, that exhibit very different physiologic properties. For example, central obesity is associated with substantial risk of insulin resistance and cardiovascular disease, while peripheral fat appears to be associated with an anti-atherogenic effect [12,13]. Differences in the biologic properties of cells within these depots have also been described [14 16]. However, these analyses have generally tended to consider subcutaneous adipose tissue as a homogeneous depot, despite evidence that different subcutaneous adipose depots exhibit a differential content of fatty acids [17], different blood vessel densities [18] and differences in insulin sensitivity [19], suggesting
Correspondence to: John K. Fraser PhD, 3020 Callan Rd, San Diego, CA 92121, USA. E-mail: [email protected] – 2007 ISCT
DOI: 10.1080/14653240701358460
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that there is heterogeneity in the biology of different subcutaneous fat depots. The majority of the research using human ASC has been performed using subcutaneous adipose tissue collected during an elective cosmetic liposuction procedure. However, these studies have failed to evaluate whether or not different subcutaneous adipose tissue depots yield different numbers or types of stem and progenitor cells. In the present study we addressed this by using clonal assays to quantify adipose-derived stem and progenitor cells in different subcutaneous adipose tissue depots. In order to minimize the effects of differences between individuals, this study was performed using paired specimens taken from two locations in a series of patients. We also evaluated the role of syringe and vacuum pump suction on cell yield.
Methods Human adipose tissue was obtained from 10 subjects undergoing elective liposuction following informed consent using an independent Institutional Review Boardapproved protocol (Independent Review Consulting, San Anselmo, CA, USA). Liposuction was performed under general anesthesia by infiltrating a ratio of 1 1.5mL of lactated Ringers solution (supplemented with epinephrine) for each milliliter of tissue to be aspirated. Tissue was removed from two subcutaneous sites, the abdomen and the hips, from all patients. Further, tissue was aspirated from each site of each patient using two approaches: (1) manual aspiration into a syringe using a 4.6-cannula and manual force, and (2) mechanical suction pump-assisted aspiration using a 4-mm or 6-mm cannula (Table 1). To avoid bias, the order of manual aspiration and mechanically assisted suction was randomized and similar volumes were harvested by each method. A summary of donor information is shown in Table 1. Immediately following collection, the tissue collection vessel was sealed and transported to the laboratory at ambient temperature. On receipt, a small sample was fixed in formalin and embedded in paraffin for histology. The tissue was washed extensively with warm (378C) saline to remove residual blood and free lipid. A small sample was fixed in buffered formalin and embedded in paraffin for histologic evaluation using hematoxylin and eosin (H&E) staining. The remaining tissue was then digested with Celase I (a proprietary mixture of clostridial collagenase and a neutral protease; Cytori Therapeutics Inc., San Diego, CA, USA) at 378C for 20 30 min. After digestion,
the adipocytes were separated by flotation, washed and counted. Similarly, the non-buoyant, adipocyte-depleted population was washed, concentrated by centrifugation at 400 g for 5 min, and counted. This fraction is referred to as adipose tissue-derived cells (ADC) in order to distinguish this heterogeneous non-cultured population from the multipotent adherent population of adipose-derived stem cells (ASC) derived by culturing ADC.
Cell counting Cell counting of adipocytes and adipocyte-depleted ADC was performed using a standard hemocytometer chamber using a live dead fluorescent dye mixture (0.3 mg/mL acridine orange, Invitrogen, Carlsbad, CA, USA; 1 mg/mL ethidium bromide, Sigma, St Louis, MO, USA) that stained nucleated cells and distinguished live cells from dead cells. Data are presented as cells per unit volume of aspirated tissue.
Fibroblast-like cell colony-forming unit (CFU-F) assay ADC were plated at two cell concentrations (1,000 and 100 cells/cm2) in triplicate in six-well plates (Corning, NY, USA) and cultured in DMEM/F-12/10% FCS/1% antibiotic antimycotic solution for 14 days, with a change of medium twice weekly. At 14 days the plates were washed with PBS, fixed with formalin and stained with hematoxylin. Colonies consisting of more than c. 50 cells were defined as CFU-F. The number of CFU-F was counted in each well and the average number of CFU-F from the triplicate sample was calculated. In order to minimize miscounting as a result of colony overlap at higher densities, only those wells containing fewer than 50 colonies were counted.
Osteoprogenitor cell assay (CFU-AP) ADC were plated in triplicate at two cell concentrations (1000 and 100 cells/cm2) in six-well plates. Osteogenic differentiation was induced by culturing cells for 3 weeks in osteogenic medium consisting of a-MEM (CellGro Mediatech, Herndon, VA, USA) and 10% FBS/1% antibiotic antimycotic solution supplemented with 0.1 mM dexamethasone (Sigma), 10 mM b-glycerophosphate (Sigma) and 50 mM L-ascorbic acid 2-phosphate (Sigma). After 1 week, the culture medium was changed twice a week with osteogenic medium without dexamethasone. After 3 weeks of culture, all wells were rinsed with PBS and stained with 1% Naphthol AS-BI phosphate and 1 mg/mL
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Table 1. Donor summary Donor ID
Gender
Age
1
F
35
Abdomen Hip
6 6
22
2
F
65
Abdomen Hip
6 6
23
3
F
45
Abdomen Hip
6 6
30
4
F
65
Abdomen Hip
4 4
22
5
F
44
Abdomen Hip
6 6
30
6
F
48
Abdomen Hip
6 6
28
7
F
37
Abdomen Hip
4 4
26
8
M
39
Abdomen Hip
4 4
26
9
F
58
Abdomen Hip
4 6
26
10
F
47
Abdomen Hip
6 6
35
Fast red TR (both from Sigma) for 20 min at 378C. After staining, the cells were washed with PBS and fixed with formalin for 15 min. Colonies consisting of more than c. 50 cells with positive alkaline phosphatase staining were defined as CFU-AP (alkaline phosphatase-positive colony-forming units). The number of CFU-AP was counted in each well at the plating density selected as above and the average number of CFU-AP from the triplicate sample was calculated. To confirm osteogenic differentiation, some wells were stained for calcium mineral deposition using a von Kossa stain.
Liposuction cannula size
BMI
mycotic solution. Adipogenic differentiation was induced after 1 week by culturing cells for 2 weeks in the same medium supplemented with 1 mM dexamethasone (Sigma), 10 mM insulin, 200 mm indomethacin and 0.5 mM IBMX (3isobutyl-1-methylxanthine). A medium change was performed twice a week. For Oil red O staining, cells were rinsed with PBS, fixed with 10% formal calcium, rinsed with 70% ethanol, stained with 2% Oil red O and counterstained with hematoxylin. Colonies consisting of more than c. 50 cells with positive Oil red O staining were defined as CFUAd. The number of CFU-Ad was counted in each well at the plating density selected as above and the average number of CFU-Ad from the triplicate sample was calculated.
Pre-adipocyte assay (adipocytic colony-forming unit; CFU-Ad) ADC were plated in triplicate at two cell concentrations (1000 and 100 cells/cm2) in six-well plates and cultured for 1 week in DMEM/F-12/10% FCS/1% antibiotic anti-
Statistical analysis Cell number, yield and colony frequency (colonies/ thousand cells plated) were evaluated using the Wilcoxon
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matched-pairs signed-ranks test. This paired analysis evaluates the extent and direction of differences between two tissue collection sites or methods within each donor. Thus each donor was effectively her or his own control. For evaluation of the harvest method, each donor had two pairs of data points; one for each donor site. For evaluation of donor site, we analyzed harvest methods separately to avoid any confounding effects of the harvest method. For determination of the statistical significance of ratios, we used the single-sample t-test and assumed the null hypothesis (no difference) ratio 1.0.
Table 2. Adipocyte yield
Donor ID
Donor site
1
Abdomen Hip
5.4 105 3.9 105
2.6 104 2.2 105
2
Abdomen Hip
8.1 104 2.7 105
7.1 104 8.5 104
3
Abdomen Hip
2.0 105 1.9 105
1.6 105 1.1 105
4
Abdomen Hip
5.3 104 4.7 104
6.4 103 3.9 104
5
Abdomen Hip
2.8 105 5.3 104
1.4 105 3.2 105
6
Abdomen Hip
2.9 105 1.8 105
1.3 105 4.2 104
7
Abdomen Hip
2.5 105 1.4 105
8.7 104 1.3 104
8
Abdomen Hip
6.2 104 8.3 104
6.1 104 7.5 104
9
Abdomen Hip
1.1 105 5.6 104
8.6 104 2.9 104
10
Abdomen Hip
1.0 105 6.4 104
5.3 104 1.8 104
Results Tissue collection Forty paired samples (20 aspirated manually, 10 hip and 10 abdominal; 20 with mechanical assistance, 10 hip and 10 abdominal) were obtained from the 10 donors. The average volume of tissue aspirated was 56 mL (range 15 150 mL, median 50 mL, SD 27 mL). There was considerable donor-to-donor and site-to-site variation in adipocyte yield with both harvest methods (Table 2). However, by harvesting each site by each method we were able to control for this variability in a manner that effectively made each site its own control. We found that the syringe-based harvest yielded significantly more adipocytes per unit volume of tissue than pumpassisted suction (Wilcoxon signed rank test P B0.001). The ratio of adipocyte yield using syringe-based suction to pump-assisted suction for each site showed considerable variation (0.2 20.2-fold); however, the mean ratio was 3.6fold (SD 4.7, single-sample t -test P B0.011). Collection using manual or mechanical suction had no significant effect on the yield (P 0.59) or viability (P 0.37) of nucleated cells in the non-buoyant fraction. Harvest method had no significant effect on the frequency of colony-forming cells within this population (CFU-F Wilcoxon signed rank test P 0.69, mean ratio syringe to vacuum pump 1.2; CFU-AP Wilcoxon P 0.08, mean ratio 0.93; CFU-Ad Wilcoxon P 0.54, mean ratio 2.0).
Effect of donor site on adipocyte yield Because of the effect of vacuum pump assistance on adipocyte yield, evaluation of the effect of donor site on adipocyte yield was limited to only those samples obtained using manual aspiration. Using this data set, abdominal tissue yielded, on average, 1.7-fold more adipocytes per
Harvest method Syringe Vacuum pump
unit volume of tissue than tissue from the hips (SD 1.3, Wilcoxon signed rank test P B0.03).
Effect of donor site on nucleated cell yield in the non-buoyant (ADC) fraction We found no significant difference in ADC yield between the two harvest methods. Tissue extracted from the hips tended to yield more ADC per unit volume than that extracted from the abdominal region. However, this effect was small (the average ratio was only 1.1-fold) and was statistically significant only for tissue harvested by vacuum pump assistance (P B0.04), not for tissue harvested by syringe (P 0.23).
Effect of donor site on colony-forming cell yield Progenitor cell yield is a function of both the yield of nucleated cells and the frequency of progenitor cells
Stem cell yield in subcutaneous adipose
within the population. For this reason we evaluated progenitor cell frequency as an independent variable. Contamination during tissue culture prevented analysis of samples from two donors, thereby limiting the sample number to eight.
Pre-adipocyte (CFU-Ad) yield Subcutaneous adipose tissue from both sites contained adipogenic colony-forming cells at a frequency of 0.8 colonies/100 cells plated (range 0 2.0). The frequency of CFU-Ad was not affected by harvest method (mean frequency in all sites using syringe-assisted suction 0.7%; mean for pump-assisted 0.8%; P B0.54 Wilcoxon signed rank). Similarly, there was no difference in the frequency of CFU-Ad between tissue aspirated from the hip and the abdomen (mean frequency in hip samples 0.78%; mean for abdomen 0.73%; Wilcoxon signed rank test P B0.58).
Osteoprogenitor cell (CFU-AP) yield Adipose tissue from the subcutaneous abdominal region contained alkaline phosphatase-positive colony-forming cells at an average frequency of 0.5 colonies/100 cells plated (range 0 1.3). In contrast, tissue aspirated from the hip contained CFU-AP at an average frequency of 2.7 colonies/100 cells plated (range 0 10). Paired analysis for each donor showed that, on average, samples from the hip had a CFU-AP frequency that was 4.3-fold higher than that from abdominal tissue using syringe aspiration (Wilcoxon signed rank test P B0.05) and a 12.8-fold higher frequency in samples harvested by pump aspiration (Wilcoxon signed rank test P B0.015). This difference can be appreciated visually in the images in Figure 1. This increased frequency translated to a statistically significant difference in the yield of CFU-AP (colonies per unit volume of tissue aspirated) between the two sites. Thus, on average, hip tissue yielded 7-fold more CFU-AP than tissue obtained from the abdominal region (Wilcoxon signed rank P B0.04). There was no effect of harvest method on CFU-AP yield or frequency.
Stem cell (CFU-F) yield Subcutaneous adipose tissue from the abdominal area contained CFU-F at an average frequency of 1.4 colonies/ 100 cells plated (range 0.6 5.0), compared with an average of 1.9 colonies/100 cells plated (range 0.6 5.0) for tissue harvested from the hip. Paired analysis indicated that this
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was statistically significant, i.e. on average samples from the hip had a CFU-F frequency that was 1.5-fold higher than that from abdominal tissue using syringe aspiration (Wilcoxon signed rank P B0.05) and a 1.5-fold higher frequency in samples harvested by pump aspiration (P B 0.05). This increased frequency translated to a 2.2-fold difference in the total yield of CFU-F (colonies per unit volume of tissue aspirated) between the two sites, although this only reached statistical significance for tissue aspirated using the vacuum pump (Wilcoxon signed rank P B0.05) and not for that harvested by syringe (Wilcoxon signed rank P B0.16).
Discussion The discovery that adipose tissue contains a population of multipotent cells [1,2] has led to the use of adipose tissuederived cells in pre-clinical models of bone and cartilage repair [20 22], skeletal muscle damage [23] and stroke [24,25]. Indeed, adipose-derived cells have been applied clinically in bone repair [26] and fistula healing [27,28]. Thus there is utility in evaluating the role of harvest method and site on the content of cells with regenerative potential within the ADC population. We found that, while mechanical pump-assisted liposuction clearly has a deleterious effect on adipocytes within the aspirated population, it has a minimal effect on other cell types, including clonogenic progenitor cells. This probably reflects the size of mature adipocytes and their consequent sensitivity to shear forces encountered during aspiration, and is consistent with earlier studies showing decreased adipocyte viability with application of a higher suction force [29] and with the commonly preferred approach when harvesting morsels of adipose tissue for free fat grafting [30]. The much smaller size of other cell types within adipose probably protects them from injury during harvest. Anecdotal data from the Cytori laboratory suggests that ultrasound-assisted liposuction is associated with substantial impairment in colony-forming cell yield (data not shown). Averaging all samples, we obtained a mean of 147 5009 32 000 ADC/mL adipose tissue. This is essentially identical to an earlier study by Hauner & Entenmann [31] evaluating tissue aspirated by syringe from the femoral and abdominal regions of 24 obese women: a mean of 135 00097900 cells/g. Further, we found that the frequency of clonogenic progenitor cells within this population was considerably greater than that present in
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Figure 1. Abdominal adipose tissue yields a lower frequency of osteoprogenitor cells (CFU-AP). Equal numbers of ADC (1000 and 100 cells/cm2) from different sites were cultured for 3 weeks in osteogenic medium and then stained for alkaline phosphatase expression. Images of three representative donors.
Stem cell yield in subcutaneous adipose
other sources of cells with similar properties. In particular, the frequency of CFU-F and CFU-AP in primary BM aspirates has been estimated at 1 in 10 000 to 1 in 1 million, although this depends upon the age of the donor and the presence of morbidities such as osteoporosis and rheumatoid arthritis [32 35]. In contrast, we found that the frequency of cells capable of giving rise to colonies that appear similar to CFU-F and CFU-AP in digested lipoaspirate was in the order of 1 in 100. This is in agreement with our earlier work [36] and with that of others [37]. Combining the frequency of CFU-F within the ADC population and the number of ADC retrieved per unit volume of tissue we calculate that, on average, 1 mL of subcutaneous adipose tissue yields approximately 1500 2000 CFU-F. We also found that, while the frequency of clonogenic cells was not significantly impacted by the harvest method, there were significant differences between different donors and between different sites within the same donor. This study was not designed or powered to evaluate the effects of donor variables such as age, gender, body mass index (BMI), history of weight gain or loss, etc., that might explain the donor-to-donor variation. Rather, our intent was to evaluate the impact of donor site by comparing the frequency of clonogenic cells obtained from one site with that obtained from a second site of the same donor and then, by applying the Wilcoxon sign rank test, to determine whether the direction and extent of any difference between sites was consistent across the 10 donors tested. This approach showed that there was no significant difference in the frequency of adipogenic progenitor cells between the hips and the abdomen. In contrast, we found that tissue collected from the hip contained a c. 1.5-fold higher frequency of CFU-F and 4.3 12.8-fold higher frequency of CFU-AP. The reason for the higher frequency of CFU-AP is not clear. Alkaline phosphatase is a widely used, although not absolutely specific, marker for osteogenesis. Thus it is possible that the alkaline phosphatase-positive colonies observed were not truly osteogenic. However, we confirmed the osteogenic nature of these cultures by showing positive von Kossa staining after 3 weeks in osteogenic medium. Further, the frequency of CFU-AP observed in the present study (0.5% for abdominal tissue, 2.7% for hip tissue) is similar to that described by Mitchell et al. [37] and lower than the frequency these authors describe for
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Alizarin red-positive colonies derived from digested adipose tissue (6.3%). It is possible that these differences reflect differences in the ratio of deep and superficial subcutaneous adipose tissue collected from the two sites. Deep subcutaneous adipose tissue has been shown to contain a higher frequency of blood vessels than the superficial layer [18]. It is possible that the CFU-AP assay applied here recognizes blood vessel pericytes, a population that has previously been shown to possess an osteoprecursor-like phenotype [38,39], and that the higher CFU-AP frequency is simply a reflection of increased pericyte content. In this regard it is interesting to note that CFU-F share certain features with pericytes and vascular smooth muscle progenitor cells [40]. However, pericytes have also been shown to possess an adipogenic potential [41,42] and, hence, a finding of increased CFU-AP content because of pericyte cross-reactivity would be expected to be associated with an increased frequency of adipogenic progenitors. We did not detect a harvest site-related difference in adipogenic progenitors. In summary, our data demonstrate that digestion of adipose tissue yields clonogenic progenitor cells at a very high frequency compared with marrow and that, within the limits of the present study, the method of harvest has little impact on the content of such cells. We also demonstrate that different subcutaneous sites within the same individual can yield populations with significantly different contents of clonogenic progenitor cells. Thus the location from which the tissue is to be harvested should be considered in potential clinical applications of adiposederived stem and progenitor cells, and the hip may be a preferred site for the harvest of cells for regenerative medicine.
Acknowledgements This study was funded entirely by Cytori Therapeutics Inc. Financial disclosure: JKF and MZ are shareholders in, and JKF, MZ, IW, and ZA receive financial compensation from, Cytori Therapeutics Inc. EW has no financial conflicts of interest with this study.
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Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots JK Fraser1, I Wulur1, Z Alfonso1, M Zhu1 and ES Wheeler2 1
Cytori Therapeutics Inc., San Diego, and 2Aesthetic Plastic Surgery Center, La Mesa, California, USA
Background Human adipose tissue has been shown to contain multipotent cells with properties similar to mesenchymal stromal cells. While there have been many studies of the biology of these cells, no study has yet evaluated issues associated with tissue harvest. Methods Adipose tissue was obtained from the subcutaneous space of the abdomen and hips of 10 donors using both syringe and pump-assisted liposuction. Tissue was digested with collagenase and then assayed for the presence of different stem and progenitor cell types using clonogenic culture assays, including fibroblast colony-forming unit (CFU-F) and alkaline phosphatase-positive colony-forming unit (CFU-AP). Paired analysis of samples obtained from the same individual was used to compare harvest method and site.
adipocyte-depleted fraction. There was considerable donor-to-donor variation in stem cell recovery. However, paired analysis of tissue obtained from different subcutaneous sites in the same donor showed that tissue harvested from the hip yielded 2.3-fold more CFU-F/unit volume and a 7-fold higher frequency of CFU-AP than that obtained from the abdomen. These differences were statistically significant. Discussion Harvest site influences the stem and progenitor cell content of subcutaneous adipose tissue. Keywords adipose adult stem cell, liposuction, subcutaneous depot.
Results Syringe suction provided significantly greater recovery of adipocytes and a non-significant trend towards improved recovery of cells in the
Introduction In recent years it has become evident that adipose tissue contains a population of stem and progenitor cells with differentiation capacity beyond adipogenesis [1]. For example, different groups have demonstrated the ability of adipose tissue-derived stem cells (ASC) to differentiate in vivo or in vitro into cells exhibiting features characteristic of osteoblasts, chondroblasts, skeletal myoblasts [2,3], cardiac myocytes [4], neuronal cells [5,6], hepatocytes [7], hematopoietic cells [8] and endothelial cells [9,10]. We and others have shown that at least some of these different lineages can be derived from single multipotent cells, indicating the presence of a novel stem cell population within adipose tissue [2,11].
Adipose tissue depots are generally separated into two categories, subcutaneous (or peripheral) adipose and visceral (or central) adipose, that exhibit very different physiologic properties. For example, central obesity is associated with substantial risk of insulin resistance and cardiovascular disease, while peripheral fat appears to be associated with an anti-atherogenic effect [12,13]. Differences in the biologic properties of cells within these depots have also been described [14 16]. However, these analyses have generally tended to consider subcutaneous adipose tissue as a homogeneous depot, despite evidence that different subcutaneous adipose depots exhibit a differential content of fatty acids [17], different blood vessel densities [18] and differences in insulin sensitivity [19], suggesting
Correspondence to: John K. Fraser PhD, 3020 Callan Rd, San Diego, CA 92121, USA. E-mail: [email protected] – 2007 ISCT
DOI: 10.1080/14653240701358460
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that there is heterogeneity in the biology of different subcutaneous fat depots. The majority of the research using human ASC has been performed using subcutaneous adipose tissue collected during an elective cosmetic liposuction procedure. However, these studies have failed to evaluate whether or not different subcutaneous adipose tissue depots yield different numbers or types of stem and progenitor cells. In the present study we addressed this by using clonal assays to quantify adipose-derived stem and progenitor cells in different subcutaneous adipose tissue depots. In order to minimize the effects of differences between individuals, this study was performed using paired specimens taken from two locations in a series of patients. We also evaluated the role of syringe and vacuum pump suction on cell yield.
Methods Human adipose tissue was obtained from 10 subjects undergoing elective liposuction following informed consent using an independent Institutional Review Boardapproved protocol (Independent Review Consulting, San Anselmo, CA, USA). Liposuction was performed under general anesthesia by infiltrating a ratio of 1 1.5mL of lactated Ringers solution (supplemented with epinephrine) for each milliliter of tissue to be aspirated. Tissue was removed from two subcutaneous sites, the abdomen and the hips, from all patients. Further, tissue was aspirated from each site of each patient using two approaches: (1) manual aspiration into a syringe using a 4.6-cannula and manual force, and (2) mechanical suction pump-assisted aspiration using a 4-mm or 6-mm cannula (Table 1). To avoid bias, the order of manual aspiration and mechanically assisted suction was randomized and similar volumes were harvested by each method. A summary of donor information is shown in Table 1. Immediately following collection, the tissue collection vessel was sealed and transported to the laboratory at ambient temperature. On receipt, a small sample was fixed in formalin and embedded in paraffin for histology. The tissue was washed extensively with warm (378C) saline to remove residual blood and free lipid. A small sample was fixed in buffered formalin and embedded in paraffin for histologic evaluation using hematoxylin and eosin (H&E) staining. The remaining tissue was then digested with Celase I (a proprietary mixture of clostridial collagenase and a neutral protease; Cytori Therapeutics Inc., San Diego, CA, USA) at 378C for 20 30 min. After digestion,
the adipocytes were separated by flotation, washed and counted. Similarly, the non-buoyant, adipocyte-depleted population was washed, concentrated by centrifugation at 400 g for 5 min, and counted. This fraction is referred to as adipose tissue-derived cells (ADC) in order to distinguish this heterogeneous non-cultured population from the multipotent adherent population of adipose-derived stem cells (ASC) derived by culturing ADC.
Cell counting Cell counting of adipocytes and adipocyte-depleted ADC was performed using a standard hemocytometer chamber using a live dead fluorescent dye mixture (0.3 mg/mL acridine orange, Invitrogen, Carlsbad, CA, USA; 1 mg/mL ethidium bromide, Sigma, St Louis, MO, USA) that stained nucleated cells and distinguished live cells from dead cells. Data are presented as cells per unit volume of aspirated tissue.
Fibroblast-like cell colony-forming unit (CFU-F) assay ADC were plated at two cell concentrations (1,000 and 100 cells/cm2) in triplicate in six-well plates (Corning, NY, USA) and cultured in DMEM/F-12/10% FCS/1% antibiotic antimycotic solution for 14 days, with a change of medium twice weekly. At 14 days the plates were washed with PBS, fixed with formalin and stained with hematoxylin. Colonies consisting of more than c. 50 cells were defined as CFU-F. The number of CFU-F was counted in each well and the average number of CFU-F from the triplicate sample was calculated. In order to minimize miscounting as a result of colony overlap at higher densities, only those wells containing fewer than 50 colonies were counted.
Osteoprogenitor cell assay (CFU-AP) ADC were plated in triplicate at two cell concentrations (1000 and 100 cells/cm2) in six-well plates. Osteogenic differentiation was induced by culturing cells for 3 weeks in osteogenic medium consisting of a-MEM (CellGro Mediatech, Herndon, VA, USA) and 10% FBS/1% antibiotic antimycotic solution supplemented with 0.1 mM dexamethasone (Sigma), 10 mM b-glycerophosphate (Sigma) and 50 mM L-ascorbic acid 2-phosphate (Sigma). After 1 week, the culture medium was changed twice a week with osteogenic medium without dexamethasone. After 3 weeks of culture, all wells were rinsed with PBS and stained with 1% Naphthol AS-BI phosphate and 1 mg/mL
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Table 1. Donor summary Donor ID
Gender
Age
1
F
35
Abdomen Hip
6 6
22
2
F
65
Abdomen Hip
6 6
23
3
F
45
Abdomen Hip
6 6
30
4
F
65
Abdomen Hip
4 4
22
5
F
44
Abdomen Hip
6 6
30
6
F
48
Abdomen Hip
6 6
28
7
F
37
Abdomen Hip
4 4
26
8
M
39
Abdomen Hip
4 4
26
9
F
58
Abdomen Hip
4 6
26
10
F
47
Abdomen Hip
6 6
35
Fast red TR (both from Sigma) for 20 min at 378C. After staining, the cells were washed with PBS and fixed with formalin for 15 min. Colonies consisting of more than c. 50 cells with positive alkaline phosphatase staining were defined as CFU-AP (alkaline phosphatase-positive colony-forming units). The number of CFU-AP was counted in each well at the plating density selected as above and the average number of CFU-AP from the triplicate sample was calculated. To confirm osteogenic differentiation, some wells were stained for calcium mineral deposition using a von Kossa stain.
Liposuction cannula size
BMI
mycotic solution. Adipogenic differentiation was induced after 1 week by culturing cells for 2 weeks in the same medium supplemented with 1 mM dexamethasone (Sigma), 10 mM insulin, 200 mm indomethacin and 0.5 mM IBMX (3isobutyl-1-methylxanthine). A medium change was performed twice a week. For Oil red O staining, cells were rinsed with PBS, fixed with 10% formal calcium, rinsed with 70% ethanol, stained with 2% Oil red O and counterstained with hematoxylin. Colonies consisting of more than c. 50 cells with positive Oil red O staining were defined as CFUAd. The number of CFU-Ad was counted in each well at the plating density selected as above and the average number of CFU-Ad from the triplicate sample was calculated.
Pre-adipocyte assay (adipocytic colony-forming unit; CFU-Ad) ADC were plated in triplicate at two cell concentrations (1000 and 100 cells/cm2) in six-well plates and cultured for 1 week in DMEM/F-12/10% FCS/1% antibiotic anti-
Statistical analysis Cell number, yield and colony frequency (colonies/ thousand cells plated) were evaluated using the Wilcoxon
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matched-pairs signed-ranks test. This paired analysis evaluates the extent and direction of differences between two tissue collection sites or methods within each donor. Thus each donor was effectively her or his own control. For evaluation of the harvest method, each donor had two pairs of data points; one for each donor site. For evaluation of donor site, we analyzed harvest methods separately to avoid any confounding effects of the harvest method. For determination of the statistical significance of ratios, we used the single-sample t-test and assumed the null hypothesis (no difference) ratio 1.0.
Table 2. Adipocyte yield
Donor ID
Donor site
1
Abdomen Hip
5.4 105 3.9 105
2.6 104 2.2 105
2
Abdomen Hip
8.1 104 2.7 105
7.1 104 8.5 104
3
Abdomen Hip
2.0 105 1.9 105
1.6 105 1.1 105
4
Abdomen Hip
5.3 104 4.7 104
6.4 103 3.9 104
5
Abdomen Hip
2.8 105 5.3 104
1.4 105 3.2 105
6
Abdomen Hip
2.9 105 1.8 105
1.3 105 4.2 104
7
Abdomen Hip
2.5 105 1.4 105
8.7 104 1.3 104
8
Abdomen Hip
6.2 104 8.3 104
6.1 104 7.5 104
9
Abdomen Hip
1.1 105 5.6 104
8.6 104 2.9 104
10
Abdomen Hip
1.0 105 6.4 104
5.3 104 1.8 104
Results Tissue collection Forty paired samples (20 aspirated manually, 10 hip and 10 abdominal; 20 with mechanical assistance, 10 hip and 10 abdominal) were obtained from the 10 donors. The average volume of tissue aspirated was 56 mL (range 15 150 mL, median 50 mL, SD 27 mL). There was considerable donor-to-donor and site-to-site variation in adipocyte yield with both harvest methods (Table 2). However, by harvesting each site by each method we were able to control for this variability in a manner that effectively made each site its own control. We found that the syringe-based harvest yielded significantly more adipocytes per unit volume of tissue than pumpassisted suction (Wilcoxon signed rank test P B0.001). The ratio of adipocyte yield using syringe-based suction to pump-assisted suction for each site showed considerable variation (0.2 20.2-fold); however, the mean ratio was 3.6fold (SD 4.7, single-sample t -test P B0.011). Collection using manual or mechanical suction had no significant effect on the yield (P 0.59) or viability (P 0.37) of nucleated cells in the non-buoyant fraction. Harvest method had no significant effect on the frequency of colony-forming cells within this population (CFU-F Wilcoxon signed rank test P 0.69, mean ratio syringe to vacuum pump 1.2; CFU-AP Wilcoxon P 0.08, mean ratio 0.93; CFU-Ad Wilcoxon P 0.54, mean ratio 2.0).
Effect of donor site on adipocyte yield Because of the effect of vacuum pump assistance on adipocyte yield, evaluation of the effect of donor site on adipocyte yield was limited to only those samples obtained using manual aspiration. Using this data set, abdominal tissue yielded, on average, 1.7-fold more adipocytes per
Harvest method Syringe Vacuum pump
unit volume of tissue than tissue from the hips (SD 1.3, Wilcoxon signed rank test P B0.03).
Effect of donor site on nucleated cell yield in the non-buoyant (ADC) fraction We found no significant difference in ADC yield between the two harvest methods. Tissue extracted from the hips tended to yield more ADC per unit volume than that extracted from the abdominal region. However, this effect was small (the average ratio was only 1.1-fold) and was statistically significant only for tissue harvested by vacuum pump assistance (P B0.04), not for tissue harvested by syringe (P 0.23).
Effect of donor site on colony-forming cell yield Progenitor cell yield is a function of both the yield of nucleated cells and the frequency of progenitor cells
Stem cell yield in subcutaneous adipose
within the population. For this reason we evaluated progenitor cell frequency as an independent variable. Contamination during tissue culture prevented analysis of samples from two donors, thereby limiting the sample number to eight.
Pre-adipocyte (CFU-Ad) yield Subcutaneous adipose tissue from both sites contained adipogenic colony-forming cells at a frequency of 0.8 colonies/100 cells plated (range 0 2.0). The frequency of CFU-Ad was not affected by harvest method (mean frequency in all sites using syringe-assisted suction 0.7%; mean for pump-assisted 0.8%; P B0.54 Wilcoxon signed rank). Similarly, there was no difference in the frequency of CFU-Ad between tissue aspirated from the hip and the abdomen (mean frequency in hip samples 0.78%; mean for abdomen 0.73%; Wilcoxon signed rank test P B0.58).
Osteoprogenitor cell (CFU-AP) yield Adipose tissue from the subcutaneous abdominal region contained alkaline phosphatase-positive colony-forming cells at an average frequency of 0.5 colonies/100 cells plated (range 0 1.3). In contrast, tissue aspirated from the hip contained CFU-AP at an average frequency of 2.7 colonies/100 cells plated (range 0 10). Paired analysis for each donor showed that, on average, samples from the hip had a CFU-AP frequency that was 4.3-fold higher than that from abdominal tissue using syringe aspiration (Wilcoxon signed rank test P B0.05) and a 12.8-fold higher frequency in samples harvested by pump aspiration (Wilcoxon signed rank test P B0.015). This difference can be appreciated visually in the images in Figure 1. This increased frequency translated to a statistically significant difference in the yield of CFU-AP (colonies per unit volume of tissue aspirated) between the two sites. Thus, on average, hip tissue yielded 7-fold more CFU-AP than tissue obtained from the abdominal region (Wilcoxon signed rank P B0.04). There was no effect of harvest method on CFU-AP yield or frequency.
Stem cell (CFU-F) yield Subcutaneous adipose tissue from the abdominal area contained CFU-F at an average frequency of 1.4 colonies/ 100 cells plated (range 0.6 5.0), compared with an average of 1.9 colonies/100 cells plated (range 0.6 5.0) for tissue harvested from the hip. Paired analysis indicated that this
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was statistically significant, i.e. on average samples from the hip had a CFU-F frequency that was 1.5-fold higher than that from abdominal tissue using syringe aspiration (Wilcoxon signed rank P B0.05) and a 1.5-fold higher frequency in samples harvested by pump aspiration (P B 0.05). This increased frequency translated to a 2.2-fold difference in the total yield of CFU-F (colonies per unit volume of tissue aspirated) between the two sites, although this only reached statistical significance for tissue aspirated using the vacuum pump (Wilcoxon signed rank P B0.05) and not for that harvested by syringe (Wilcoxon signed rank P B0.16).
Discussion The discovery that adipose tissue contains a population of multipotent cells [1,2] has led to the use of adipose tissuederived cells in pre-clinical models of bone and cartilage repair [20 22], skeletal muscle damage [23] and stroke [24,25]. Indeed, adipose-derived cells have been applied clinically in bone repair [26] and fistula healing [27,28]. Thus there is utility in evaluating the role of harvest method and site on the content of cells with regenerative potential within the ADC population. We found that, while mechanical pump-assisted liposuction clearly has a deleterious effect on adipocytes within the aspirated population, it has a minimal effect on other cell types, including clonogenic progenitor cells. This probably reflects the size of mature adipocytes and their consequent sensitivity to shear forces encountered during aspiration, and is consistent with earlier studies showing decreased adipocyte viability with application of a higher suction force [29] and with the commonly preferred approach when harvesting morsels of adipose tissue for free fat grafting [30]. The much smaller size of other cell types within adipose probably protects them from injury during harvest. Anecdotal data from the Cytori laboratory suggests that ultrasound-assisted liposuction is associated with substantial impairment in colony-forming cell yield (data not shown). Averaging all samples, we obtained a mean of 147 5009 32 000 ADC/mL adipose tissue. This is essentially identical to an earlier study by Hauner & Entenmann [31] evaluating tissue aspirated by syringe from the femoral and abdominal regions of 24 obese women: a mean of 135 00097900 cells/g. Further, we found that the frequency of clonogenic progenitor cells within this population was considerably greater than that present in
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Figure 1. Abdominal adipose tissue yields a lower frequency of osteoprogenitor cells (CFU-AP). Equal numbers of ADC (1000 and 100 cells/cm2) from different sites were cultured for 3 weeks in osteogenic medium and then stained for alkaline phosphatase expression. Images of three representative donors.
Stem cell yield in subcutaneous adipose
other sources of cells with similar properties. In particular, the frequency of CFU-F and CFU-AP in primary BM aspirates has been estimated at 1 in 10 000 to 1 in 1 million, although this depends upon the age of the donor and the presence of morbidities such as osteoporosis and rheumatoid arthritis [32 35]. In contrast, we found that the frequency of cells capable of giving rise to colonies that appear similar to CFU-F and CFU-AP in digested lipoaspirate was in the order of 1 in 100. This is in agreement with our earlier work [36] and with that of others [37]. Combining the frequency of CFU-F within the ADC population and the number of ADC retrieved per unit volume of tissue we calculate that, on average, 1 mL of subcutaneous adipose tissue yields approximately 1500 2000 CFU-F. We also found that, while the frequency of clonogenic cells was not significantly impacted by the harvest method, there were significant differences between different donors and between different sites within the same donor. This study was not designed or powered to evaluate the effects of donor variables such as age, gender, body mass index (BMI), history of weight gain or loss, etc., that might explain the donor-to-donor variation. Rather, our intent was to evaluate the impact of donor site by comparing the frequency of clonogenic cells obtained from one site with that obtained from a second site of the same donor and then, by applying the Wilcoxon sign rank test, to determine whether the direction and extent of any difference between sites was consistent across the 10 donors tested. This approach showed that there was no significant difference in the frequency of adipogenic progenitor cells between the hips and the abdomen. In contrast, we found that tissue collected from the hip contained a c. 1.5-fold higher frequency of CFU-F and 4.3 12.8-fold higher frequency of CFU-AP. The reason for the higher frequency of CFU-AP is not clear. Alkaline phosphatase is a widely used, although not absolutely specific, marker for osteogenesis. Thus it is possible that the alkaline phosphatase-positive colonies observed were not truly osteogenic. However, we confirmed the osteogenic nature of these cultures by showing positive von Kossa staining after 3 weeks in osteogenic medium. Further, the frequency of CFU-AP observed in the present study (0.5% for abdominal tissue, 2.7% for hip tissue) is similar to that described by Mitchell et al. [37] and lower than the frequency these authors describe for
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Alizarin red-positive colonies derived from digested adipose tissue (6.3%). It is possible that these differences reflect differences in the ratio of deep and superficial subcutaneous adipose tissue collected from the two sites. Deep subcutaneous adipose tissue has been shown to contain a higher frequency of blood vessels than the superficial layer [18]. It is possible that the CFU-AP assay applied here recognizes blood vessel pericytes, a population that has previously been shown to possess an osteoprecursor-like phenotype [38,39], and that the higher CFU-AP frequency is simply a reflection of increased pericyte content. In this regard it is interesting to note that CFU-F share certain features with pericytes and vascular smooth muscle progenitor cells [40]. However, pericytes have also been shown to possess an adipogenic potential [41,42] and, hence, a finding of increased CFU-AP content because of pericyte cross-reactivity would be expected to be associated with an increased frequency of adipogenic progenitors. We did not detect a harvest site-related difference in adipogenic progenitors. In summary, our data demonstrate that digestion of adipose tissue yields clonogenic progenitor cells at a very high frequency compared with marrow and that, within the limits of the present study, the method of harvest has little impact on the content of such cells. We also demonstrate that different subcutaneous sites within the same individual can yield populations with significantly different contents of clonogenic progenitor cells. Thus the location from which the tissue is to be harvested should be considered in potential clinical applications of adiposederived stem and progenitor cells, and the hip may be a preferred site for the harvest of cells for regenerative medicine.
Acknowledgements This study was funded entirely by Cytori Therapeutics Inc. Financial disclosure: JKF and MZ are shareholders in, and JKF, MZ, IW, and ZA receive financial compensation from, Cytori Therapeutics Inc. EW has no financial conflicts of interest with this study.
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Doherty MJ, Ashton BA, Walsh S et al . Vascular pericytes express osteogenic potential in vitro and in vivo . J Bone Miner Res 1998;13:828 38. Lamagna C, Bergers G. The bone marrow constitutes a reservoir of pericyte progenitors. J Leukoc Biol 2006;80:677 81. Iyama K, Ohzono K, Usuku G. Electron microscopical studies on the genesis of white adipocytes: differentiation of immature pericytes into adipocytes in transplanted preadipose tissue. Virchows Arch B Cell Pathol Incl Mol Pathol 1979;31:143 55. Cinti S, Cigolini M, Bosello O et al . A morphological study of the adipocyte precursor. J Submicrosc Cytol 1984;16:243 51.