Clonal analysis reveals remarkable functional heterogeneity during hematopoietic stem cell emergence

Clonal analysis reveals remarkable functional heterogeneity during hematopoietic stem cell emergence

In conventional opinion, hematopoietic stem cells (HSCs) are alike, possessing robust self-renewal and multilineage differentiation capacity. However, growing evidence has revealed striking functional heterogeneity among individual HSCs, particularly in the aspect of lymphomyeloid output [1][2][3]. Four subtypes of HSCs with distinct differentiation patterns have been identified and designated as α-, β-, γ-, and δ-HSCs [4]. Only lymphoid-deficient (α) and lymphomyeloid-balanced (β) HSCs bear durable engraftment potential and extensive self-renewal activity [5]. Although differentiation program of individual HSCs can be stably self-propagated, occasional occurrence of inter-conversions among programs has also been observed [5]. HSC heterogeneity exhibits developmental stage-related fluctuation, especially regarding the prevalence of α-HSCs, being minor in fetal liver but high in aging bone marrow (BM) [5]. The arising concept of HSC heterogeneity leads to a series of key questions, as to its embryonic origin, regulatory mechanisms, and relationship with leukemogenesis and therapeutics.
The first transplantable adult-like HSCs become detectable in the aorta-gonad-mesonephros (AGM) region of mice around embryonic day (E) 10.5 [6]. At this time, the AGM region contains at least two types of pre-HSCs [7], characterized by a high expression level of CD201 [8]. Pre-HSCs possess abundant expression of endothelial signature genes and can develop into bona fide HSCs after co-culture with stromal cells and cytokines in vitro. It is of great interest but remains unknown whether and to what extent HSC heterogeneity presents when pre-HSCs and HSCs emerge in mid-gestation embryos.
Approximately 3 AGM regions at E11.0 (40-44 somite pairs, sp) contain only one mature HSC. This frequency markedly increases to one HSC per AGM at E11.5 (45-48 sp) [9]. To ensure rigorous clonal analysis, we dissected E11 AGM tissues, with each AGM equally divided into 2 (E11.0) or 3 (E11.5) aliquots for subsequent direct transplantation into lethally irradiated adult mice. Re-cipients demonstrating ≥ 1% donor-derived white blood cells (WBCs) in peripheral blood after a minimum of 4 months were considered to be long-term reconstituted (Supplementary information, Figure S1A). Out of 139 recipients, 54 demonstrated successful long-term (> 4 months) reconstitution. 14 recipients repopulated by both aliquots from 7 E11.0 embryos were ruled out, and the remaining 40 were used for further analyses (Supplementary information, Figure S1B). The results are highly reminiscent of previous data using limiting dilution assay [9,10], ensuring the reconstitution at a clonal level.
Similar to what was observed in adult BM [4], embryonic β-HSCs demonstrated a significantly higher re-Cell Research (2017) 27:1065-1068. www.nature.com/cr constitution ability than γ-HSCs. Moreover, B-dominant γ-HSCs displayed a remarkably lower reconstitution ability than balanced γ-HSCs ( Figure 1F and Supplementary information, Figure S2B). Unlike balanced γ-HSCs showing a sustained reconstitution, B-dominant γ-HSCs manifested decreased reconstitution levels over time ( Figure 1F). These data suggest that the most limited repopulating potential of HSCs during midgestation is related to their compromised ability to differentiate into myeloid and T lymphoid lineages.
In adult BM, β-HSCs display extensive self-renewal activity, whereas γ-HSCs fail to repopulate secondary recipients [4,5]. We performed secondary transplantations using 3 primary recipients of β-HSCs and 5 primary recipients of balanced γ-HSCs. Cells from the 3 primary recipients of β-HSCs reconstituted 9 of 18 secondary recipients (Supplementary information, Figure S2C-S2D). Unexpectedly, only 1 secondary recipient displayed a β-HSC differentiation pattern, whereas the other 8 showed a balanced γ-HSC differentiation pattern (Figure 1G and Supplementary information, Figure S2D). Interestingly, cells from 4 out of 5 primary recipients of balanced γ-HSCs reconstituted 13 of 23 secondary recipients (Supplementary information, Figure S2C-S2D). Among them, 11 maintained the balanced γ-HSC pattern, and the other 2 showed a B-dominant γ-HSC feature, highly suggesting preservation of differentiation programs of balanced γ-HSCs ( Figure 1G and Supplementary information, Figure S2D).Thus, embryonic β-HSCs can give rise to both β-and γ-HSCs, and the embryonic γ-HSCs can only give rise to γ-HSCs, supporting a higher hierarchy of β-HSCs than that of γ-HSCs, in line with what was observed in BM [4,5]. In contrast to adult BM [4,5], E11 AGM regions contain predominantly γ-HSCs but less β-HSCs. Moreover, embryonic balanced γ-HSCs showed a much stronger self-renewal potential than those in adults, whereas embryonic β-HSCs showed a limited preservation of their differentiation pattern upon secondary transplantation.
Finally, we analyzed a recently reported dataset of singe-cell RNA-seq of multiple HSC-competent populations [8]. Out of 77 hematopoietic lineage-differentiation genes, 36 genes showed significantly differential expression between embryonic (pre-HSCs and fetal liver HSCs) and adult populations, most of which (29/36) displayed higher expression levels in the former (Supplementary information, Figure S2H). Among these 29 genes, 17 are related to lymphoid lineage differentiation and the expression level distributions of these genes in the fetal liver HSCs were usually either like those in pre-HSCs or similar to those in BM HSCs, suggesting a gradual change of the differentiation programs along with development ( Figure 1K). In summary, our study unveils a heterogeneity in pre-HSC/HSC composition in E11 mouse embryos, indicating that functional heterogeneity in HSCs exists from the very beginning of embryonic HSC emergence and persists throughout the whole lifespan [4,5] ( Figure  1L). This study reveals at least two major HSC subtypes closely associated with the wall of large arteries during the course of HSC emergence, prior to the colonization of mature HSCs to fetal liver. The mechanism for the absence of α-HSCs deserves further investigations, which might be related to the influence of the microenvironment [5]. In addition, we are searching for candidate surface markers that can be used to isolate certain subtypes of pre-HSCs and HSCs in various hemogenic and hematopoietic niches.