Purification and characterization of human adipose-resident microvascular endothelial progenitor cells

Flow cytometric analysis of the SVF

Freshly isolated SVF samples contained 3.84 ± 1.43 × 10⁵ cells per gram of adipose tissue (n = 9). The SVF could be segregated into four primary cell populations, AEPCs (CD45⁻CD34⁺CD31⁺), ASCs (CD45⁻CD34⁺CD31⁻), hematopoietic cells (CD45⁺), and other cell types (CD45⁻CD34⁻; Fig. 1B). The proportions of hematopoietic cells, AEPCs, ASCs, and other cells in the SVF were 41.1 ± 10.0%, 25.2 ± 6.8%, 21.0 ± 2.8%, and 12.7 ± 4.7%, respectively. Fresh AEPC population was further analyzed for other surface markers and characterized as CD45⁻CD31⁺CD34⁺CD105⁺CD146⁺CD157^±CD200^±. The AEPC population could be further divided into two subpopulations, CD31^highCD34^high and CD31^lowCD34^low. The CD31^highCD34^high AEPC population showed high expression levels of CD157 and CD200, whereas the CD31^lowCD34^low AEPC population presented various fluorescent intensities for CD157 and CD200 expression (Fig. 1B). Fresh ASC population was characterized as CD45⁻CD31⁻CD34⁺CD105⁻CD146⁻CD157^±CD200⁻. CD105 is a well-known mesenchymal stem cell marker, and ASCs expressed CD105 in vitro after adhering to the plastic surfaces of culture plates (Fig. 4).

Purification and expansion of AEPCs

As a preliminary experiment, we attempted to extract the SVF using three enzyme solutions: #1: collagenase and CaCl₂; #2: collagenase, CaCl₂, and DNase1; #3: collagenase, CaCl₂, DNase1, and Pol188. The percentage of AEPCs in the SVF tended to be higher when extracted using enzyme solution #2 (Supplemental Fig. S1A). We also examined the effects of various collagenase concentrations (0.0–2.0%) in enzyme solution #2. The numbers of total nucleated cells and ASCs in the SVF increased in a dose-dependent manner with increasing collagenase concentrations. The numbers of AEPCs in the SVF peaked at collagenase concentrations of 0.1–0.4% (Supplemental Fig. S1B). Based on these results, the same volume of the #2 enzyme formulation containing 0.2% (w/v) collagenase, 3 mM CaCl₂, 1000 U/mL DNase1, and HBSS was added to adipose tissue in subsequent experiments.

We also examined the use of several good manufacturing practice (GMP)-grade enzymes and combinations to prepare purified AEPCs for clinical use (Supplemental Fig. S1C).

The SVF was separated by MACS using CD45 and CD31 microbeads, both CD45⁻CD31⁺ (AEPC-rich) and CD45⁻CD31⁻ (ASC-rich) MACS-separated fractions were obtained, and the expression profiles of CD45, CD34, and CD31 were analyzed by flow cytometry (Fig. 2A). Population analysis showed that the CD45⁻CD31⁺ (AEPC-rich) fraction contained 84.3 ± 5.8% of AEPCs (CD45⁻CD34⁺CD31⁺ cells), leukocytes (CD45⁺ cells) at 0.9 ± 0.3%, ASCs (CD45⁻CD34⁺CD31⁻ cells) at 4.7 ± 2.3%, and others (CD45⁻CD34⁻ cells and debris) at 10.1 ± 5.0% (n = 5) (Fig. 2A). The SVF, CD45⁻CD31⁺ fraction, and CD45⁻CD31⁻ fraction were cultured for 10 days in EGM-2 media, and photographs were obtained daily from days 2 to 10 using a phase-contrast microscope, with representative photographs shown in Fig. 2B (SVF), Fig. 2C (CD45⁻CD31⁺ fraction) and 2D (CD45⁻CD31⁻ fraction). AEPCs (cobblestone shape) and ASCs (spindle shape) could be morphologically discriminated under the microscope. In AEPC-rich culture (CD45⁻CD31⁺ fraction), AEPC colonies (dotted line) were observed, and fewer ASCs were observed compared with the unenriched SVF culture until day 7 (Fig. 2B,C). However, even after enrichment, AEPC colonies were eventually overwhelmed by proliferating ASCs starting on day 10 (Fig. 2C), indicating that a single MACS enrichment step was insufficient for AEPC purification and expansion. Flow cytometric analyses indicated that the ASC proportion in the CD45⁻CD31⁺ (AEPC-rich) fraction increased after 5 days of culture (Supplemental Fig. S2).

MACS separation and culture of the SVF. (A) The freshly isolated, hemolyzed SVF was separated into CD45⁻CD31⁺ (AEPC-rich) and CD45⁻CD31⁻ fractions (ASC-rich) in the first MACS separation. During this flow cytometric analysis, lymphocyte gating events in the SSC versus SFC plot were set to 100%. CD45⁻CD34⁺CD31⁺ (magenta ROI) was defined as the AEPC population. CD45⁻CD34⁺CD31⁻ (violet ROI) was defined as the ASC population. Bar charts display the ratio between the CD45⁻CD31⁺CD34⁺ and CD45⁻CD31⁺CD34⁻ populations in the SVF (upper panels), CD45⁻CD31⁺, (middle panels), and CD45⁻CD31⁻ fractions (lower panels), which were 0.54 to 0.46 ± 0.075 (n = 9, 9 donors, mean ± SD), 0.95 to 0.05 ± 0.03 (n = 5, 5 donors, mean ± SD), and 0.18 to 0.82 ± 0.08 (n = 5, 5 donors, mean ± SD), respectively. Cultivation of the (B) freshly isolated SVF, (C) first MACS-separated CD45⁻CD31⁺ fraction (AEPC-rich), and (D) first MACS-separated CD45⁻CD31⁻ fraction (ASC-rich). The morphologies of all the populations were photographed daily from days 2–10 using a phase-contrast microscope (Leica DM IL LED with a camera MC170HD; 100× magnification). Bars represent 100 µm. All the cell populations were seeded at 1 × 10⁴ nucleated cells/0.2 mL of EGM-2 media/cm². Dotted circles indicate AEPC colonies. The experiment was performed twice, independently from 2 donors.

Therefore, we performed a second MACS enrichment using CD31 microbeads to further purify the AEPC population. A schematic view of the procedure of AEPC purification is shown in Fig. 3A. After performing purification steps of fresh SVF (Fig. 3Ba) and the first MACS CD45⁻CD31⁺ (AEPC-rich) fraction (Fig. 3Bb), the second MACS separation step of cultured AEPC-rich populations was performed after either 4.5 days (before the timing of ASC proportion increase) or 7 days (after the timing of ASC proportion increase) of culture (Fig. 3Bc,d,g,h). When separated on day 4.5 (steps in Fig. 3Bc,d), the respective percentages of CD45⁻CD31⁺ cells before and after the second MACS separation were 91.6% and 97.1%, respectively (n = 2; Fig. 3C). When separated on day 7 (steps in Fig. 3Bg,h), the respective percentages of CD45⁻CD31⁺ cells before and after separation were 70.4% and 94.7%, respectively (n = 2; Fig. 3C). AEPCs separated on day 4.5 were cultured for 6 days (Fig. 3Be,f) and expanded with satisfactory purity through at least 5 passages (Fig. 3D). By contrast, AEPCs separated on day 7 were overwhelmed by proliferating ASCs after 6 days of culture (Fig. 3Bi,j).

Establishment of the AEPC purification. (A) Schematic view of the AEPC purification method from human lipoaspirates. AEPCs were purified from the SVF using two MACS sorting steps (CD45⁻CD31⁺ sorting and only CD31⁺ sorting) and adherent cultures. (B,C) Optimization of the timing for the second MACS sorting step for AEPC purification. Cells were dissociated from the cell culture surface with TrypLE Express Enzyme after either 4.5 or 7 days of culture and were subjected to MACS separation using CD31 microbeads. Cell surface markers (CD45, CD31, and CD34) were examined by flow cytometry. The percentage of CD45⁻CD31⁺ (AEPC) cells in CD45⁻CD31⁻ (ASCs, isotype⁺ cells, other cells, and debris) cells is shown in magenta. ASCs drastically decreased CD34 expression in vitro, which could be observed in the CD45⁻CD31⁻ population (arrowheads). Scale bars in microscopic photographs represent 100 µm. This experiment was performed twice, independently from 2 donors. (D) Morphologies of purified AEPCs over time. The photographs were taken using a phase-contrast microscope (Leica DM IL LED with a camera MC170HD; 100× magnification). P1D6 indicates the culture period at passage 1, after 6 days in culture. The experiment was performed twice, independently from 2 donors. Bars represent 100 µm.

Time-dependent changes in CD marker expression profiles AEPC and ASC cultures

AEPCs (obtained as the CD45⁻CD31⁺ fraction after two MACS separation steps) and ASCs (obtained as the CD45⁻CD31⁻ fraction after the first MACS separation) were cultured, and time-dependent changes in CD marker expression profiles (CD45, CD31, CD34, CD146, and CD105) were examined. Cultured AEPCs maintained CD31, CD146, and CD105 expression through at least 5 passages, whereas CD34 expression decreased over time in culture (Figs. 1B and 4). ASCs began to express CD105 after seeding, despite being CD105⁻ before seeding, and maintained expression through at least 5 passages, although CD34 expression decreased over time in culture. CD146 expression was not observed in ASCs.

Time-dependent changes in AEPC cell morphologies and surface marker expression profiles. (A) The CD45⁻CD31⁺ and (B) CD45⁻CD31⁻ MACS-separated fractions were characterized for the expression of CD markers, including CD45, CD31, CD34, CD146, and CD105. The experiment was performed twice, independently from 2 donors. The graph indicates 2 donors, depicted by red and blue lines. The X-axis (passage number) indicates that the CD45⁻CD31⁺ fraction at P5 corresponded to dissociated AEPCs at P4D4, as shown in Fig. 3D.

Characterization of expanded AEPCs

We characterized and performed functional analyses of expanded AEPCs (passage 5), compared against expanded HUVECs (passage 5), using immunocytochemistry, CFU-EC, EC network formation, and flow cytometry analyses. Immunocytochemistry showed similar CD31 and vWF expression profiles and isolectin-B4 binding capacity between AEPCs and HUVECs (Fig. 5A). CD31 expression was detected in 99.8 ± 0.4% and 99.3 ± 1.1% of AEPCs and HUVECs, respectively. vWF was expressed in 90.3 ± 3.3% and 76.4 ± 8.0% of AEPCs and HUVECs, respectively. Both AEPCs and HUVECs were 100% positive for isolectin-B4 binding capacity.

Characterization and functional analysis of expanded AEPCs compared with HUVECs. All the experiments were performed using AEPCs and HUVECs at passage number 5. (A) Fluorescent immunocytochemistry of the expression profiles of endothelial cell-specific markers, CD31, vWF, and the binding capacity of isolectin-B4, in AEPCs and HUVECs. Nuclear staining was assessed by 4′,6-diamidino-2-phenylindole (DAPI). Photographs were taken using a confocal microscope (OLYMPUS, FV1000 with a CCD camera DP71 and an objective lens UPlanFL N 40×/1.30 oil; 400× magnification) as a plane image. Bars represent 100 µm. The graphs show the percentage of endothelial marker-positive cells among DAPI-positive cells. The data represent two independent experiments (2 donors), and percentages were determined from cultivations performed in triplicate. The bars represent means ± SD (n = 6, **P < 0.01, two-tailed unpaired t-test). (B) CFU-EC analysis of AEPCs and HUVECs. Cells were seeded at 125 cells per 35-mm-diameter well (12.6 cells/cm²) and cultured for 7 days. Each experiment was performed twice independently (two donors) in technical triplicates. The bars represent means ± SD (n = 3; n.s., not significant by two-tailed unpaired t-test). (C) Network formation capacity of AEPCs compared with that of HUVECs. Representative phase-contrast pictures corresponding to the right-hand side of the extracted network skeleton. The total segment length (magenta color), total branch length (yellow–green color), and total length were determined using the Angiogenesis Analyzer plugin for ImageJ software. Scale bars represent 100 µm. The experiment was performed independently three times (3 donors) in technical pentaplicates. Bars represent means ± SD (n = 5, **P < 0.01; n.s., not significant by two-tailed unpaired t-test). (D) Cultured AEPCs and HUVECs were characterized by the expression of CD45, CD31, CD34, CD157, and CD200 by flow cytometry analysis. This experiment was performed independently three times (3 donors) for AEPCs and independently two times (2 lots, 5- or 7-donor mixture) for HUVECs and is listed in Table 2.

In the CFU-EC assay, the colony forming cells in AEPCs and HUVECs were 59.7 ± 8.4% and 47.3 ± 7.9%, respectively (which did not differ significantly) on day 7. AEPCs proliferated more slowly in colonies, and the average size of AEPC colonies was smaller than that of HUVEC colonies (Fig. 5B). Even if using the same medium, EGM-2MV, AEPCs formed colonies smaller in size than HUVECs, and reached a similar size on day 12 to that of HUVECs on day 7 (Supplemental Figure S3).

In the network formation assay, both AEPCs and HUVECs formed tube-like networks. The total length, total segment length, and total branch length were analyzed, and all the parameters were similar between AEPCs and HUVECs. The total network lengths measured for AEPCs and HUVECs were 32.7 ± 1.8 × 10³ and 32.1 ± 0.5 × 10³ pixels/field, respectively. The total segment lengths for AEPCs and HUVECs were 16.4 ± 1.5 × 10³ and 18.3 ± 1.6 × 10³ pixels/field, respectively. The total branch lengths for AEPCs and HUVECs were 15.2 ± 1.1 × 10³ and 12.5 ± 1.3 × 10³ pixels/field, respectively (Fig. 5C).

Flow cytometry indicated that expanded AEPCs expressed reduced levels of tissue-resident EPC markers, such as CD157 and CD200, during culture, and CD200 was not detectable by passage number 5. AEPCs (passage 5) contained CD157⁺ population at 1.1–2.7% among CD45⁻CD31⁺ cells (n = 3). By contrast, HUVECs (passage 5) did not express either CD157 or CD200 (Fig. 5D and Table 2).