hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding

Abstract

This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair. Moreover, hESC-secreted factors improve the viability of human cortical neurons in an Alzheimer's disease (AD) model, suggesting that these factors can enhance the maintenance and regeneration of multiple tissues in the aging body.

Mechanisms of action of hESC-secreted proteins that enhance human and mouse myogenesis

“…Proliferation was significantly enhanced and differentiation inhibited in both human and mouse muscle progenitor cells by the hESC-conditioned medium (Figure 1A, B). Analogously, hESC growth medium (mTeSR1) conditioned by hESC culture enhanced myoblast proliferation more effectively than mTeSR1 alone, though mouse myoblasts exhibited significantly enhanced proliferation in both media (Supplementary Figure 1A, B and [17]).…”

“…We recently published that in the mouse system, the pro-myogenic activity of hESC-conditioned Opti-MEM is contained in proteins with heparin-binding activity [17]. To explore the evolutionary conservation of this phenomenon in a human system, we cultured primary human muscle progenitor cells in a 50/50 mixture of the low-mitogen differentiation medium plus hESC-conditioned Opti-MEM that was either complete or depleted of heparin-binding proteins.…”

“…Considering that medium conditioned by hESCs, but not their differentiated progeny, has pro-regenerative activity (Figure 1), which is lost upon Proteinase K treatment [14, 17], we performed a comparative proteomics antibody array analysis of these two media (Materials and Methods). Replicate experiments indicated specific proteins that were more abundant in the hESC-conditioned medium compared with medium conditioned by differentiated hESCs, and the top candidates are listed in Table 1.…”

“…In addition to the canonical Smad signaling, the MAPK pathway can also be induced by BMP [15]. In adult myogenesis, BMP signaling is upregulated after satellite cell activation both in vivo and in vitro, and inhibition of BMP signaling promotes myogenic differentiation [16,17]. BMPs may promote satellite cell proliferation by activating their downstream targets, the differentiation-inhibiting Id genes, which inhibit transcription factors that promote cell differentiation [17].…”

“…Furthermore, many FGFs bind heparin [16], and we demonstrated that the heparin-binding fraction of the hESC-produced factors exert robust proliferative effects on mouse muscle progenitor cells and provide a rejuvenating stimulus for old murine muscle regeneration [17]. Here, we show that hESC-produced proteins enhance the regenerative capacity not only of mouse but also human muscle progenitor cells, determine the molecular mechanisms by which hESC-produced proteins enhance human myogenic proliferation, and establish the molecular identity of several of these clinically relevant factors.…”