HPLC profile of proteoglycans. Metabolically radiolabeled proteoglycans from normal and Hurler MAPCs were subjected to DEAE-Sephacel anion-exchange HPLC. The column was eluted at 1 mL/minute using an increasing NaCl gradient, and 1-mL fractions were collected. The ³⁵S and ³H radioactivities were measured and plotted for each fraction. The proportion of radioactivity in each peak was calculated as a percentage of the total radioactivity in the proteoglycan-containing peaks. DPM indicates disintegrations per minute.

Analysis of disaccharide composition of normal and Hurler MAPC HS. HS from normal and Hurler MAPCs was enzymatically digested and the resulting disaccharides separated by reverse-phase ion pair HPLC. Individual disaccharides were quantified by determining the area under the peaks. n = 3 separate experiments. (A) High-performance liquid chromatography (HPLC) tracing of disaccharides from normal and Hurler MAPCs from one representative experiment. Peak no. 1 (0S): UAGlCNAc; peak no. 2 (NS): UAGlcNS; peak no. 3 (6S): UAGlcNAc6S; peak no. 4 (NS, 6S): UAGlcNS6S; peak no. 5 (2S, NS): UA2SGlcNS; peak no. 6 (2S, NS, 6S): UA2SGlcNS6S. (B) Disaccharide composition of normal and Hurler HS, expressed as percent of total HS. (C) Proportions of the 3 6-sulfated disaccharides in normal and Hurler HS. (D) Total 6-sulfation in normal and Hurler HS. ^*P < .05. (E) Progressive decrease in proportion of UA-GlcNS6S with increasing total HS accumulation. Correlation coefficient (R) = 0.99. Data in panels B-D are shown as the mean ± standard error (SE).

FGF-2–induced proliferation of normal and Hurler MAPCs. Normal or Hurler MAPCs were cultured for 24 hours in absence or presence of a range of concentrations of FGF-2. The numbers of cells present in each well were determined as detailed in “Materials and methods.” Cell numbers are expressed as a ratio (number of cell in wells supplemented with FGF-2–number of cells in absence of FGF-2). (A) Proliferation of normal and Hurler MAPCs in the absence or presence of the indicated concentrations of FGF-2. In separate wells in the same experiments, normal MAPCs were cultured in presence of 50% CM from Hurler MAPCs. n = 4 to 6 independent experiments with replicates. Comparison between normal MAPCs and the other conditions: ^*P < .01, ¶P < .005, §P < .002, ^**P < .001. (B) Proliferation of heparitinase I– and III–treated normal MAPCs, in the absence or presence of 50% CM from normal or Hurler MAPCs, was examined as in panel A. n = 3 independent experiments with replicates. Comparison between proliferation seen in presence of normal versus Hurler CM: §§P < .05, ¶P < .005, §P < .002. (C) Proliferation of heparitinase I– and III–treated Hurler MAPCs, in the absence or presence of 50% CM from normal or Hurler MAPCs, was examined as in panel A. n = 3 independent experiments with replicates. Comparison between proliferation seen in presence of normal versus Hurler CM: §§P < .05, ^*P < .01, §P < .002, ^**P < .001.

FGF-2 induced proliferation of F32 cells on fixed MAPCs. F32 cells were cultured for 24 hours on fixed monolayers of normal and Hurler MAPCs in the presence of the indicated concentrations of FGF-2. The numbers of cells present in each well were determined as detailed in “Materials and methods.” Cell numbers are expressed as a ratio (number of cells in wells supplemented with FGF-2/number of cells in absence of FGF-2). Proliferation of F32 cells plated on heparitinase I and III (hep'ase)–treated normal and Hurler MAPCs was also examined in parallel wells. n = 2 independent experiments with replicates. ○ indicates Normal MAPC; •, Hurler MAPC; □, No cell layer; ▴, Hep'ase treated Hurler MAPC; and ▵, Hep'ase treated normal MAPC.

Size distribution of HS. HSs were purified separately from each proteoglycan peak that was resolved by anion-exchange HPLC, and subjected to gel filtration chromatography on a CL-6B column, as described in “Materials and methods.” Three regions containing large-, intermediate-, and small-sized molecules were identified, based on the elution profile of normal intracellular HPLC peak no. 1 HS (K_av 0.1-0.4, 0.4-0.55, and 0.55-0.9, respectively [K_av = (elution volume - void volume)/(total volume - void volume)]). The proportion of HS present in each of these regions was calculated as a percentage of the total, for HS from intracellular peak no. 2 as well as HS from HPLC peaks no. 1 and no. 2 from the ECM.

(A-C) Binding of ¹²⁵I-FGF-2 to MAPC cell-surface receptors. Binding of ¹²⁵I-FGF-2 to FGFR1 in presence of cell-surface HS. The binding of ¹²⁵I-FGF-2 to FGFR1 on normal and Hurler MAPCs was determined as described in “Materials and methods.” Briefly, normal or Hurler MAPCs were incubated with ¹²⁵I-FGF-2 to induce binding to cell-surface receptors. Cell lysates were prepared and subjected to SDS-PAGE. FGFR1 was visualized by Western immunoblotting, and ¹²⁵I-FGF-2 by autoradiography. In the control lanes, either ²⁵I-FGF-2, DSS, or both were omitted, as shown. The densitometric intensity of the band in each lane is shown below the respective lanes. (A) Western immunoblotting with anti-FGFR1 antibody, demonstrating comparable expression of FGFR1 by normal and Hurler MAPCs. (B) Autoradiograph of the same membrane for ¹²⁵I-FGF-2, demonstrating colocalization of FGFR1 and ¹²⁵I-FGF-2. (C) The extent of binding of ¹²⁵I-FGF-2 to FGFR1 determined by densitometry was expressed as a ratio of ¹²⁵I-FGF-2 to FGFR1 (B/A). (D) ¹²⁵I-FGF-2 binding to cell-surface receptors on normal and Hurler MAPCs. The binding of ¹²⁵I-FGF-2 to normal and Hurler MAPC monolayers was examined as described in “Materials and methods.” The counts per minute (cpm) bound (specific binding) were corrected for the number of cells in each well, and then expressed as the proportion of cpm bound to normal MAPC/10⁵ cells/well. Data are from 2 to 3 independent experiments with replicates. N: normal; H: Hurler; hep'ase: heparitinases I and III. Significance of differences between conditions is described in “Results.”

Apoptosis of Hurler MAPCs in presence of FGF-2. (A) Apoptosis was determined in Hurler MAPCs grown on coverslips placed in 6-well plates. Color definitions are as follows: green indicates viable cells; yellow, apoptotic cells (arrow); red, dead cells (arrowhead). Images were acquired on an Olympus BX60 fluorescent microscope using an UPlanApo 10×/0.4 dry objective lens at room temperature (final magnification, × 100). Fluorochromes were Cy3 (red) and 6-CF (green). Multiple fields were photographed using an Olympus U-ULS100HG digital camera system and SPOT 2.12 software (Diagnostics Instruments). Red and green images were merged and the composite figure made using Adobe Photoshop CS software. (i) Normal MAPCs grown on coverslips in standard MAPC medium (containing serum, EGF, and PDGF-BB). (ii) Hurler MAPCs grown on coverslips in standard MAPC medium (as in panel i). (iii) Hurler MAPCs grown on coverslips in FGF-2–supplemented serum-free medium, in presence of Hurler MAPCs in Transwells (TW) for 5 days. (iv) Hurler MAPCs grown on coverslips in FGF-2–supplemented serum-free medium, in presence of normal MAPCs in Transwells for 5 days. (B) Apoptotic and dead cells on the coverslips in the lower chambers of wells were counted on the indicated days after changing to FGF-2–supplemented serum-free medium and placement of Transwells with either Hurler or normal MAPCs. Data in the columns show the percentages of apoptotic and dead cells (upper and lower sections of the columns, respectively). The total height of the columns indicates the percentages of apoptotic and dead cells. The upper and lower error bars indicate the standard error (SE) of the mean for apoptotic and dead cells, respectively. Comparison between wells with Hurler MAPCs in the Transwells versus wells with normal MAPCs in Transwells: ^*P < .02 for apoptotic cells; ^**P < .05 for apoptotic and dead cells.