Stable Contribution of Lineage-Restricted Stem Cells to Steady-State Aged Human Hematopoiesis

Background

Multipotent self-renewing hematopoietic stem cells (HSCs) possess the potential to replenish all mature blood cell lineages, playing a critical role in safeguarding life-long replenishment of millions of short-lived blood cells every second during steady-state hematopoiesis and in response to hematopoietic challenges. Our knowledge of the functional properties and roles of mammalian HSCs is largely based on studies in mice. Studies of steady-state replenishment of blood cell lineages by individual human HSC clones have been hampered by the large number of active HSCs in adult hematopoiesis. Moreover, while the dynamic contribution of HSC clones to different lineages can only be assessed by tracing the same individual HSC clones over time, steady-state contribution from individual HSCs has only been investigated at a single time point, both in mice and in humans.

Results

We assessed blood lineage contribution for 51 driver and 10 non-driver mutations in clonal hematopoiesis related genes, derived from 33 healthy aged volunteers (age 70-84) with normal blood parameters, with ≥2% and ≥1% mutant cell fractions (MCFs), respectively. Of these 61 clonal mutations, 3 showed involvement exclusively in the T cell lineage, and not in any other blood cell lineages or HSCs, which is consistent with reflecting long-lived T cells. Of the remaining 58 mutations, all but one could be traced back to HSCs. These 57 HSC clones were evaluated for multilineage contribution, including platelets (P), erythrocytes (E), myeloid cells (M), and B (B) and T (T) lymphocytes. In addition to the 22 HSCs replenishing all five lineages, we identified HSCs replenishing all lineages except T lymphocytes (n=30), and all three myeloid lineages (PEM) but no lymphoid cells (n=5). No further lineage restriction patterns were reproducibly observed.

In addition to the HSCs displaying PEM- and PEMB-restricted lineage replenishment patterns, other HSC clones showed distinct PEM (and PEMB) lineage bias (contributions to all PEM lineages >5 times higher than both B and T lymphocytes). Collectively, PEMB-restricted/biased (n=26) or PEM-restricted/biased (n=12) HSC replenishment was more frequent than balanced PEMBT lineage replenishment (n=15). Clonal assessment in pro-B cells in the bone marrow revealed that the steady-state B cell contribution from individual HSC clones reflects ongoing B lymphopoiesis. The PEMBT pattern was almost exclusively observed in HSC clones with DNMT3A mutations. In contrast, PEMB- and PEM-restricted/biased HSC clones showed no significant bias for mutations in specific genes and included HSC clones marked by non-driver mutations, supporting that PEMB- and PEM-restriction/biases occur independently of the mutations.

Analysis of 22 HSC clones through serially collected bone marrow samples from 11 healthy individuals up to 41 months after the first analysis revealed that the observed patterns of lineage replenishment were remarkably stable over time, whether representing balanced PEMBT, PEMB-restricted/biased, or PEM-restricted/biased HSCs. This stability was also observed upon transplantation into immune-deficient mice, suggesting that these patterns are HSC intrinsically programmed.

Phylogenetic analysis using single colony whole-genome sequencing of hematopoietic stem/progenitor cells combined with lineage contribution patterns of the identified clades in 7 donors revealed novel insights into the hierarchical relationships of HSC clones with different lineage patterns, in that PEMBT HSC clones gave rise to PEMB clones which could give rise to PEM HSC clones, but not vice versa, establishing a unidirectional hierarchical relationship between increasingly lineage-restricted/biased HSC clones.

Conclusions

Utilizing somatically acquired mutations as natural barcodes, we revealed the existence of distinct human HSC clones with stable multilineage as well as myeloid-restricted and -biased contributions to steady-state human hematopoiesis. Whether replenishing hematopoiesis in a balanced multilineage or lineage-restricted manner, individual HSCs invariably showed a remarkably stable lineage contribution pattern and clonal size over years of observation. Phylogenetic analysis demonstrated that aging leads to myeloid-biased and even myeloid-restricted production from HSCs that originally replenished hematopoiesis in a fully multipotent manner.