The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture‐expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand‐receptor complex formation. We previously reported on an FGF2‐interacting HS variant (termed HS2) isolated from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here, we detail the isolation of an FGF2 affinity‐purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP‐2 or VEGF165. This process used a peptide sequence derived from the heparin‐binding domain of FGF2 as a substrate to affinity‐isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF‐BB, or VEGF165. Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long‐term supplementation of cultures with HS8 increased both hMSC numbers and their colony‐forming efficiency without adversely affecting the expression of hMSC‐related cell surface antigens. This strategy further exemplifies the utility of affinity‐purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture‐expansion of hMSCs destined for cellular therapy. J. Cell. Physiol. 232: 566–575, 2017. © 2016 Wiley Periodicals, Inc. We here report a novel strategy for ex vivo expansion of human mesenchymal stem cells that replies on supplementation of growth factor‐binding heparan sulfates, instead of the growth factor itself into the culture. The advantage of such approach is to avoid the reported adverse effect of FGF2 on MSCs upon long‐term exogenous supplementation, and more importantly, the quality of hMSCs expanded is not compromised after long‐term culture. The work is of particular importance given the wide therapeutic use of hMSCs and the key role of FGF2 in the technology of hMSC scaling‐up.