Right on schedule: mimicking developmental signal timing improves in vitro blood progenitor differentiation

Current Limitations and Challenges of In Vitro-Derived Hematopoietic Stem/Progenitor Cells

Human pluripotent stem cells (hPSCs) can theoretically give rise to all blood cell types, offering promising applications in disease modeling, drug screening, and cell therapies. However, a limited understanding of the signaling pathways that specify human hematopoietic stem/progenitor cells (HSPCs)—which sit at the apex of blood formation—hinders their effective use in research and clinical applications.

"In vitro-derived HSPCs exhibit cell-surface markers similar to those of their in vivo counterparts. However, significant transcriptional—and, more importantly, functional—differences remain between these two sources of hematopoietic progenitors. Strategies that address these transcriptomic discrepancies may offer a promising way to expand the clinical applicability of in vitro-derived hematopoietic stem cells," said Professor Mo Li from the Biological and Environmental Science and Engineering Division at King Abdullah University of Science and Technology (KAUST), who co-led the study with Professor Juan Carlos Izpisua Belmonte at Altos Labs.

Transcriptomic Differences Between in vivo- and in vitro-derived HSPCs

Professor Li and his colleagues used RNA sequencing to compare the transcriptomes of in vivo- and in vitro-derived HSPCs. They found that genes associated with the WNT signaling pathway are significantly downregulated in in vivo HSPCs compared to their in vitro counterparts, indicating that reducing WNT signaling after the hemogenic endothelium (HE) stage is crucial for HSPC maturation. “Notably, this WNT downregulation during HSPC development is observed both in mice and humans,” he explained. “This cross-species consistency suggests that the phenomenon is inherent rather than an artifact of culture conditions.”

The Role of Late-Stage WNT Inhibition

To further investigate the regulatory role of WNT signnaling, the team used small-molecule inhibitors and genetically modified pluripotent stem cells to modulate the pathway and its downstream effectors, respectively, during late-stage hematopoietic differentiation. Their results showed that inhibiting WNT signaling promotes HSPC generation in vitro and leads to improved myeloid chimerism in immuncompromised mice. These findings suggest that late-stage WNT inhibition not only remodels their transcriptome to more closely resemble bona fide hematopoietic stem cells but also enhances HSPC differentiation and function.

Intrinsic Deficiencies in In Vitro-Derived HSPCs

Further investigation revealed that failure to downregulate WNT signaling in in vitro-derived HSPCs causes dysregulation of HOX genes, premature expression of lineage-specific genes, abnormally increased mitochondrial activity, and altered chromatin accessibility to hematopoietic transcription factors. These intrinsic deficiencies distinguish hiPSC-derived progenitor cells from endogenous HSCs. partial rescue of these defects is achievable through WNT pathway inhibition, and WNT inhibition partially corrects these defects.

This study identifies a potential strategy to enhance the differentiation and functionality of hPSC-derived HSPCs, bringing them closer to their in vivo counterparts and improving their therapeutic potential.