Fig 2: Effect of TTX-030 and POM-1 on the pattern of3HATP metabolism by SK-MEL-28 cells. A, Radio-TLC assays tracked the inhibition of CD39-mediated hydrolysis of3H-labeled ATP of SK-MEL-28 cells by POM-1 (10 µM) and a suboptimal treatment of TTX-030 (200 ng/ml). The kinetic parameters (Vmax and Km) for all catalytic activities were calculated from the presented curves and summarized in the right panel. Error bars, S.E., n= 3. *P < .05 as compared with control. B, Cultured SK-MEL-28 cells were incubated with 100 μM3HATP and subsamples of the medium were collected at timed intervals, followed by TLC separation and quantification of the amount of3HATP and its dephosphorylated3H-metabolites, ADP, AMP and adenosine (ADO). The ordinate shows the relative content of3H-labeled nucleotides and ADO expressed as percentage of total radioactivity added. Error bars, S.E., n= 2. Lower panel depicts a representative autoradiographic image of an overall pattern of metabolism of3HATP by control and treated cells. The blank (Bl) shows the radiochemical purity of3HATP in the absence of cells. Arrows indicate the positions of nucleotide and ADO standards

Fig 3: Comparison of the ability of different fibroblast subpopulations to support epidermal growth on DED. (a) Isolation of fibroblast subpopulations by flow cytometry. (b–f) Hematoxylin and eosin staining (scale bars = 100 μm) and (g–k) immunofluorescence staining (scale bars = 200 μm) of DED organotypic cultures (b, g) without fibroblasts or (c, h) seeded with unfractionated (lin–CD90+) fibroblasts, (d, i) CD90+CD39+ (enriched in papillary) fibroblasts, (e, j) CD90+CD39– (depleted of papillary) fibroblasts, and (f, k) CD90+CD36+ (reticular/pre-adipocyte) fibroblasts. Keratin 14 (green) marks keratinocytes, and vimentin (white) marks mesenchymal cells (fibroblasts). Experiments were repeated for a minimum of two biological replicates (donor cells derived from female [age 19 years] breast skin and female [age 37 years] breast skin; DED derived from male [age 47 years] abdominal skin, and representative images are displayed. In the case of the CD90+CD39+ cells, two of three experiments involved additional selection for CD26– cells to further enrich for the papillary cell population (CD90+CD39+CD26–). (l) Quantification of epidermal thickness, (m) density of fibroblasts within 300 μm of the epidermis, and (n) relative abundance of fibroblasts at different depths from the epidermis. adipo, adipocyte; DED, decellularized human dermis; FB, fibroblasts; FSC, forward scatter detector; K14, keratin 14; lin–, lineage negative; ns, not significant; SSC, side scatter detector.

Fig 4: Human dermal fibroblast subpopulations maintain functional differences in vitro. (a, b) Expression of LUM and COL6A5 is enriched in CD90+ population compared with an unfractionated dermal cell suspension. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three replicates. (c) CD39 expression is detectable in primary CD31–CD45–ECad– cells but is lost after a single passage in culture (d). However, expression of (d, e) CD90 and (e) CD36 is retained. (f) lin–CD90+CD39+ and lin–CD90+CD36+ cells exhibit morphological differences in vitro, there is considerable intersample variation. Breast skin donor age, 19 years; abdominal skin donor age, 43 years; and thigh skin donor age, 46 years. Scale bars = 50 μm). (g) Quantitative PCR showing retention of LUM and loss of COL6A5 in culture. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. (h–j) Expression of genes associated with (h) Wnt signaling, (i) inflammation and immunity, and (j) ECM remodeling. Gene expression is normalized to 18S and is expressed as mean ± standard deviation for three biological replicates derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. (k–o) Modulation of expression of cell surface markers in response to IFN-γ stimulation in culture (blue, CD39+IFN-γ; yellow, CD36+IFN-γ; red, unstained control). Top row: representative flow plots. Bottom row: quantitation of data from three independent experiments derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001. ECM, extracellular matrix; FSC, forward scatter detector; lin–, lineage negative; MFI, mean fluorescence intensity; SSC, side scatter detector.

Fig 5: TTX-030 is an uncompetitive inhibitor of CD39 ATPase activity. A, SK-MEL-28 CD39-mediated ATPase initial velocities of Pi release as a function of ATP concentration were plotted for a titration of TTX-030 IgG (left panel) or Fab (right panel) and globally fit to the partial, mixed inhibition equation to obtain the inhibitor constant, Ki, α, and β. α is a parameter that determines the mechanism of inhibition where α < 1 indicates an uncompetitive mechanism; β is a parameter that determines the extend of partial inhibition where zero indicates complete inhibition and 1.0 indicates no inhibition. IgG, Error bars, S.D., n= 4. Fab, Error bars, S.E., n= 1. B, Binding kinetics of a highly related precursor to TTX-030 to recombinant extracellular domain of human CD39, rhCD39-ECD, in the presence or absence of non-hydrolyzable ATP analog, AMP-PNP. Binding to rhCD39-ECD was approximately 2-fold stronger in the presence of 1 mM AMP-PNP, supporting an uncompetitive mechanism. Data were globally fit to 1:1 binding model. KD, dissociation constant; kon, association (or on) rate; koff, dissociation (or off) rate