Ex Vivo Expansion of Human Hematopoietic Stem Cells By Mimicking Intrinsic and Extrinsic Factors in Adult Bone Marrow

The limited yield and availability of human hematopoietic stem cells (HSCs) continue to constrain life-saving HSC transplantations in clinical settings. For decades, ex vivo expansion of human HSCs has been the holy grail of hematology, yet it has proven to be challenging. Although recent advances have facilitated ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs), the expansion of serial transplantable long-term (LT) HSCs remains elusive. Developing new culture systems is imperative to provide high-quality and quantities of donor HSCs for clinical applications.

Most expansion protocols focus on stimulating HSC proliferation, often neglecting a crucial question: how to preserve the expanded HSCs in such a proliferation-favorable condition. In adult BM, HSCs are fine-tuned by complex intrinsic programs and extrinsic niche factors to maintain their properties including quiescence. We speculated that a culture condition recapitulating these factors would benefit HSC maintenance ex vivo. N-cadherin marks mesenchymal stromal/stem cells as the first reported HSC niche in adult BM. Our lab reported that YTHDF2 knockdown expands human HSCs. Therefore, we examined N-cadherin as an extrinsic niche factor and YTHDF2 as an intrinsic factor on their capacity to maintain HSCs ex vivo. We cultured human cord blood derived CD34⁺ HSPCs in Y13-27 (a small molecule inhibitor of YTHDF2), 3D-NcadP (a 3D peptide-hydrogel tagged with N-cadherin peptides) and their combination (named 3D-NcadP-Y) conditions for 4 days. 250 CD34⁺CD38^-CD45RA^-EPCR⁺ HSCs were then sorted and transplanted into irradiated NSG mice. The results showed that BM hCD45 chimerism and successful engraftment ratio of primary mice in the 3D-NcadP-Y group (23.5%, 7/9) and the fresh control group (62.6%, 7/8) were significantly higher than those in the Y13-27 (2.6%, 1/8) or 3D-NcadP (8.6%, 5/8) group. Strikingly, the engraftment was exclusively observed in the secondary transplanted mice in the 3D-NcadP-Y (4.3%, 6/9) and fresh groups (17.7%, 6/6). This data suggests that 3D-NcadP-Y can robustly maintain human LT-HSCs ex vivo.

We hypothesized that adding an HSC self-renew stimulating factor to the 3D-NcadP-Y condition and extending culture time would achieve greater HSC expansion. Therefore, we performed limiting dilution assay (LDA) with serial transplantation for the HSPCs cultured in 3D-NcadP-Y, UM729 (an agonist of HSC self-renewal) and their combination (named 3D-NcadP-Y-UM) conditions for 12 days. It shows that the 3D-NcadP-Y-UM condition achieved ~9-fold of ST-HSC expansion (assessed in primary LDA) and ~4-fold of LT-HSC expansion (quantified by secondary LDA) than the fresh group, the most rigorous control. Either 3D-NcadP-Y or UM729 alone failed to expand LT-HSCs, suggesting their cooperative role in HSC expansion. Importantly, we observed myeloid biased lineage distribution in the BM of the secondary transplanted mice in the UM729 group, while balanced lineage contributions were observed in the counterparts from the 3D-NcadP-Y-UM and fresh groups. In summary, we demonstrate that 3D-NcadP-Y-UM condition can expand both ST-HSCs and LT-HSCs with balanced multi-lineage potential.

We then investigated the molecular mechanisms underlying individual factors in the 3D-NcadP-Y-UM condition. m⁶A-seq analysis revealed the transcripts (IKZF1, IGF2BP3, SMAD1) directly targeted by Y13-27 mediated YTHDF2 inhibition. Flow cytometry assays uncovered that HSPCs in the 3D-NcadP-Y-UM condition showed lower levels of DNA damage, reactive oxygen species, metabolism rate, and cell cycle progression than those in the other groups. Single-cell multi-omics sequencing co-profiling gene expression and chromatin accessibility in the same cell reveals that HSCs in the 3D-NcadP-Y and 3D-NcadP-Y-UM conditions displayed higher genetic or epigenetic HSC signatures (e.g., stemscore, low-output and serial-engraftment) than the HSCs from the other groups, aligning closer with the fresh HSCs.

In this study, we have successfully developed a culture method, 3D-NcadP-Y-UM, that robustly expands both ST- and LT-HSCs while maintaining balanced multi-lineage potential. We also delineated the contributions of individual factors (3D, Y13-27, N-cadherin peptides, UM729) in our culture condition to the expansion. We are confident that our culture condition holds immense potential for future clinical applications.