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. 2019 Dec 12;25(23-24):1658–1666. doi: 10.1089/ten.tea.2019.0031

FIG. 3.

FIG. 3.

Trilineage differentiation potential of MACS-defined CD34+ adventitial cells. (A, B) Adipogenic differentiation of MACS-defined CD34+ adventitial cells in monolayer. (A) ORO staining of intracellular lipid accumulation at day 10 of differentiation. Inset depicts standard growth medium (SGM) control. (B) Adipogenic gene marker expression by qRT-PCR, including CEBPα, LPL, FABP4, and PPARγ, at sequential timepoints after adipogenic differentiation. **p < 0.01 in comparison to day 3. (C–E) Osteogenic differentiation of MACS-defined CD34+ adventitial cells in monolayer. (C) ALP activity at 5 days of differentiation. Inset depicts standard growth medium (SGM) control. (D) Alizarin Red (AR) staining of bone nodule formation at 10 days of differentiation. Inset depicts standard growth medium (SGM) control. (E) Osteogenic gene marker expression by qRT-PCR, including RUNX2, OPN, and OSX at sequential timepoints after osteogenic differentiation. *p < 0.05 and **p < 0.01 in comparison to day 0. (F–H) Chondrogenic differentiation of MACS-defined CD34+ adventitial cells in 3D micromass. (F) Alcian Blue/fast red staining of micromass cross sections after 21 days differentiation. (G) Safranin O staining of micromass cross sections after 21 days differentiation. (H) Expression of the chondrogenic differentiation marker, COL2, in micromass culture as sequential timepoints after chondrogenic differentiation. Black scale bar = 50 μM. Green scale bar = 250 μM. Representative data shown, repeated in biologic triplicate. ALP, alkaline phosphatase; CEBPα, CCAAT/enhancer binding protein α; COL2, collagen type 2; FABP4, fatty acid-binding protein 4; LPL, lipoprotein lipase; OPN, osteopontin; PPARγ, peroxisome proliferator-activated nuclear receptor γ; ORO, oil red O; OSX, osterix; qRT-PCR, quantitative real-time polymerase chain reaction; RUNX2, runt-related transcription factor 2. Color images are available online.