Human CD34/CD90 ASCs are capable of growing as sphere clusters, producing high levels of VEGF and forming capillaries

Abstract

Background: Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.

Methodology/principal findings: In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers--including CD34, CD90, CD29, CD44, CD105, and CD117--and endothelial-progenitor-cell markers--including CD34, CD90, CD44, and CD54. Interestingly, CD34(+)/CD90(+) cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34(+)/CD90(+) quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31(+)/VEGF(+)/Flk-1(+)) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.

Conclusions/significance: Our results demonstrate, for the first time, that CD34(+)/CD90(+) cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.

Figure 1. Representative flow cytometry analysis performed at day 0, 15, and 30 of culture.

A significant number of ASCs at day 0 are clearly positive for mesenchymal markers, including CD29 (89%), CD105 (12%), CD34 (33%), CD90 (52%), CD117 (10%), and for endothelial markers, including CD31 (9%), CD133 (4%), CD44 (5%), CD54 (9%), and Flk-1 (32%). At day 15 the CD34 and CD90 levels consistently increased (80%). After 30 days of culture, CD34 levels significantly decreased down to 8%, while the CD90 expression held steady at 80%. Later, cells were mainly positive for CD44 and CD54 antigens (87%). Control PE-conjugated and FITC-conjugated isotypes were negative and all cells were negative for CD45 antigens.

Figure 2. Representative flow cytometry analysis performed on CD34⁺/CD90⁺ cells at day 30 from sorting.

A significant number of ASCs are clearly positive for endothelial markers, including CD90 (97%), CD44 (90%), CD54 (90%), VEGF (70%), CD133 (18%), and Flk-1 (60%). Control PE-conjugated and FITC-conjugated isotypes were negative.

Figure 3. Cell cycle analysis performed using Hoechst 33342 and Ki67 both on CD34 negative and positive cells.

(A) Hoechst 33342 analysis performed CD34⁻ cells: G₀G₁ phase (98%), S phase (0,65%) and G₂M phase (0,50%); (B) Ki67 analysis performed CD34- cells: Ki67 (10%); (C) Hoechst 33342 analysis performed CD34⁺ cells: G₀G₁ phase (84%), S phase (5%) and G₂M phase (10%); (D) Ki67 analysis performed CD34+ cells: Ki67 (85%).

Figure 4. Side population assay.

Image of the CD34⁺ fraction representing a very small subset (2%), expressing the characteristic side-population profile.

Figure 5. Spheres formation, cytometric analysis and telomerase activity.

(A) Sphere clusters formed by CD34⁺/CD90⁺ cells in semisolid medium after 24 hours (Original Magnification×100); (B) Cytometric analysis on adherent cells for CD90 (80%) and CD34 (80%) antigens and on floating spheres for CD90 (2–3%) and CD34 (95–98%) antigens; (C) Telomerase activity of differentiated endothelial cells (ΔA = 0.160) was significantly reduced (p<0.001) respect to undifferentiated CD34⁺/CD90⁺ cells (ΔA = 0.377)

Figure 6. Adipogenic differentiation.

(A) ASCs in DMEM 10% FBS exhibit a fibroblast-like morphology (Original magnification 100×); (B) ASCs in adipogenic medium exhibit an adipocyte morphology (Original magnification 100×); (C) CD34⁺/CD90⁺ cells in DMEM 10% FBS showing negativity for adiponectin by immunohistochemistry (Original magnification 400×); (D) CD34⁺/CD90⁺ cells in adipogenic medium showing positivity for adiponectin by immunohistochemistry (Original magnification 100×); (E) CD34⁻/CD90⁻ cells in DMEM 10% FBS showing negativity for adiponectin by immunohistochemistry (Original magnification 100×); (F) CD34⁻/CD90⁻ cells in adipogenic medium showing negativity for adiponectin by immunohistochemistry (Original magnification 400×).

Figure 7. Endothelial differentiation.

(A) Image showing differentiated endothelial cells that, after 48 h in methylcellulose, acquire a different morphology comprising round cells with cytoplasmic granules and large flat cells with endothelial morphology (Original magnification 400×); (B) Image showing differentiated endothelial cells that, after day 7 in methylcellulose, formed an extensive intercellular tube networks (Original magnification 400×); Immunofluorescence images of differentiated endothelial cells showing positivity for several specific markers including (C) CD90 (Original magnification 100×); (D) VEGF (Original magnification 100×); (E) CD31 (Original magnification 100×); (F) Immunohistochemistry images of differentiated endothelial cells showing positivity for VEGF (Original magnification 100×); (G) At 140 hours, VEGF quantity was found to be of 5130 pg/ml with respect to the value of 336 pg/ml found at 12 hours; (H) At day 12, VEGF was 6331 pg/ml of medium with respect to 382 pg/ml found at day 1.

Figure 8. RT-PCR analysis.

(A) Representative figure of RT-PCR showing mRNA transcript expression of CD90, CD34, CD44, CD54, VEGF, Flk-1, on cells in DMEM 10% FBS 7, 15 and 30 days of culture; (B) PPARγ and adiponectin on cells in adipogenic medium at 7 and (C) 30 days of culture.