Prolonged cultivation enhances the stimulatory activity of hiPSC mesenchymal progenitor-derived conditioned medium

Human induced pluripotent stem cells represent a scalable source of youthful tissue progenitors and secretomes for regenerative therapies. The aim of our study was to investigate the potential of conditioned medium (CM) from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (MSCs). We also investigated whether prolonged cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the stimulatory activity of CM.

MSCs were isolated from 13 donors (age 20-90 years). CM derived from hiPSC-MPs was added to the MSC cultures and the effects on proliferation and osteogenic differentiation were examined after 14 days and 6 weeks. The stimulatory activity of hiPSC-MP-CM was compared with the activity of MSC-derived CM and with the activity of CM prepared from hiPSC-MPs pre-cultured in growth or osteogenic medium for 14 days. Comparative proteomic analysis of CM was performed to gain insight into the molecular components responsible for the stimulatory activity.

Primary bone marrow-derived MSC exhibited variability, with a tendency towards lower proliferation and tri-lineage differentiation in older donors. hiPSC-MP-CM increased the proliferation and alkaline phosphatase activity of MSC from several adult/aged donors after 14 days of continuous supplementation under osteogenic conditions. However, CM supplementation failed to improve the mineralization of MSC pellets after 6 weeks under osteogenic conditions. hiPSC-MP-CM showed greater enhancement of proliferation and ALP activity than CM derived from bone marrow-derived MSCs. Moreover, 14-day cultivation but not osteogenic pre-differentiation of hiPSC-MPs strongly enhanced CM stimulatory activity. Quantitative proteomic analysis of d14-CM revealed a distinct profile of components that formed a highly interconnected associations network with two clusters, one functionally associated with binding and organization of actin/cytoskeletal components and the other with structural constituents of the extracellular matrix, collagen, and growth factor binding. Several hub proteins were identified that were reported to have functions in cell-extracellular matrix interaction, osteogenic differentiation and development.

Our data show that hiPSC-MP-CM enhances early osteogenic differentiation of human bone marrow-derived MSCs and that prolonged cultivation of hiPSC-MPs enhances CM-stimulatory activity. Proteomic analysis of the upregulated protein components provides the basis for further optimization of hiPSC-MP-CM for bone regenerative therapies.