Evaluation of tenogenic differentiation potential of selected subpopulations of human adipose‐derived stem cells

Identification of a suitable cell source and bioactive agents guiding cell differentiationtowards tenogenic phenotype represents a prerequisite for advancement of cellâ based therapies for tendon repair. Human adiposeâ derived stem cells (hASCs) are apromising, yet intrinsically heterogenous population with diversified differentiationcapacities. In this work, we investigated antigenicallyâ defined subsets of hASCsexpressing markers related to tendon phenotype or associated with pluripotency thatmight be more prone to tenogenic differentiation, when compared to unsortedhASCs. Subpopulations positive for tenomodulin (TNMD+ hASCs) and stage specificearly antigen 4 (SSEAâ 4+ hASCs), as well as unsorted ASCs were cultured up to 21days in basic medium or media supplemented with TGFâ β3 (10 ng/ml), or GDFâ 5(50 ng/ml). Cell response was evaluated by analysis of expression of tendonâ relatedmarkers at gene level and protein level by real time RTâ PCR, western blot, and immu-nocytochemistry. A significant upregulation of scleraxis was observed for both sub-populations and unsorted hASCs in the presence of TGFâ β3. More prominentalterations in gene expression profile in response to TGFâ β3 were observed forTNMD+ hASCs. Subpopulations evidenced an increased collagen III and TNC deposi-tion in basal medium conditions in comparison with unsorted hASCs. In the particularcase of TNMD+ hASCs, GDFâ 5 seems to influence more the deposition of TNC.Within hASCs populations, discrete subsets could be distinguished offering variedsensitivity to specific biochemical stimulation leading to differential expression oftenogenic components suggesting that cell subsets may have distinctive roles in thecomplex biological responses leading to tenogenic commitment to be further exploredin cell based strategies for tendon tissues.The authors acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON 2020, under the Tendon Therapy Train Marie Skłodowska‐Curie Innovative Training Network grant agreement No. 676338, the TEAMING Grant agreement No 739572 ‐ The Discoveries CTR, and the Achilles Twinning Project No. 810850. Authors acknowledge also the European Research Council CoG MagTendon No. 772817, the FCT (Fundação para a Ciência e a Tecnologia) Project MagTT PTDC/CTM‐ CTM/29930/2017 (POCI‐01‐0145‐FEDER‐29930), and the Project NORTE‐01‐0145‐FEDER‐000021: “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marine‐derived biomaterials and stem cells”, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). ADE and JdB acknowledge the financial contribution of the Dutch province of Limburg in the LINK (FCL67723; “Limburg INvesteert in haar Kenniseconomie”) knowledge economy project