Decoding human bone marrow hematopoietic stem and progenitor cells from fetal to birth.

Bone marrow (BM) is the dominant site of hematopoiesis after 20 post-conception weeks (PCWs), but the intricacies of hematopoietic development in fetal BM up to birth and its involvement in malignancies remain unknown. Here, we compared the single-cell transcriptomic profile of BM hematopoietic stem and progenitor cells (HSPCs) at the early (12-14 PCW), middle (19-22 PCW) second trimester, and the neonatal stage. The stemness of hematopoietic stem cell and multipotent progenitor (HSC/MPP) is established at the middle second trimester, then maintained until birth. Furthermore, differentiation potentials toward three lineages are enhanced after the middle second trimester for birth, accompanied by the upregulation of aerobic metabolism. Notably, decreased stemness in HSCs/MPPs and higher interferon signals in progenitors at the early second trimester rendered the HSPCs more proximal to leukemogenesis. Collectively, our work elucidated the dynamics of fetal hematopoiesis in preparation for birth, offering valuable insights into the pathological processes underlying leukemia.

p21/Zbtb18 repress the expression of cKit to regulate the self-renewal of hematopoietic stem cells.

The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21-tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT+ HSCs exhibited increased self-renewal capacity compared to p21-tdT- HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT+ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.

The ATF4-RPS19BP1 axis modulates ribosome biogenesis to promote erythropoiesis.

ABSTRACT: Hematopoietic differentiation is controlled by intrinsic regulators and the extrinsic hematopoietic niche. Activating transcription factor 4 (ATF4) plays a crucial role in the function of fetal and adult hematopoietic stem cell maintenance. However, the precise function of ATF4 in the bone marrow (BM) niche and the mechanism by which ATF4 regulates adult hematopoiesis remain largely unknown. Here, we used 4 cell-type-specific mouse Cre lines to achieve conditional knockout of Atf4 in Cdh5+ endothelial cells, Prx1+ BM stromal cells, Osx+ osteoprogenitor cells, and Mx1+ hematopoietic cells and uncovered the role of Atf4 in niche cells and hematopoiesis. Intriguingly, depletion of Atf4 in niche cells did not affect hematopoiesis; however, Atf4-deficient hematopoietic cells exhibited erythroid differentiation defects, leading to hypoplastic anemia. Mechanistically, ATF4 mediated direct regulation of Rps19bp1 transcription, which is, in turn, involved in 40 S ribosomal subunit assembly to coordinate ribosome biogenesis and promote erythropoiesis. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, our findings highlight the indispensable role of the ATF4-RPS19BP1 axis in the regulation of erythropoiesis.

Ruxolitinib improves hematopoietic regeneration by restoring mesenchymal stromal cell function in acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation. Hematopoietic dysfunction accompanied by severe aGVHD, which may be caused by niche impairment, is a long-standing clinical problem. However, how the bone marrow (BM) niche is damaged in aGVHD hosts is poorly defined. To comprehensively address this question, we used a haplo-MHC-matched transplantation aGVHD murine model and performed single-cell RNA-Seq of nonhematopoietic BM cells. Transcriptional analysis showed that BM mesenchymal stromal cells (BMSCs) were severely affected, with a reduction in cell ratio, abnormal metabolism, compromised differentiation potential, and defective hematopoiesis-supportive function, all of which were validated by functional assays. We found that ruxolitinib, a selective JAK1/2 inhibitor, ameliorated aGVHD-related hematopoietic dysfunction through a direct effect on recipient BMSCs, resulting in improved proliferation ability, adipogenesis/osteogenesis potential, mitochondria metabolism capacity, and crosstalk with donor-derived hematopoietic stem/progenitor cells. By inhibiting the JAK2/STAT1 pathway, ruxolitinib maintained long-term improvement of aGVHD BMSC function. Additionally, ruxolitinib pretreatment in vitro primed BMSCs to better support donor-derived hematopoiesis in vivo. These observations in the murine model were validated in patient samples. Overall, our findings suggest that ruxolitinib can directly restore BMSC function via the JAK2/STAT1 pathway and, in turn, improve the hematopoietic dysfunction caused by aGVHD.

Uncovering the emergence of HSCs in the human fetal bone marrow by single-cell RNA-seq analysis.

During fetal development, human hematopoietic stem cells (HSCs) colonize the bone marrow (BM), where they self-renew and sustain hematopoiesis throughout life; however, the precise timepoint at which HSCs seed the BM is unclear. We used single-cell RNA-sequencing to map the transcriptomic landscape of human fetal BM and spleen hematopoietic stem/progenitor cells (HSPCs) and their microenvironment from 10 to 14 post-conception weeks (PCWs). We further demonstrated that functional HSCs capable of reconstituting long-term multi-lineage hematopoiesis in adult NOG mice do not emerge in the BM until 12 PCWs. In contrast, functional HSCs were not detected in the spleen by 14 PCWs. By comparing the niche-HSPC interactions between BM and spleen, we identified ligand-receptor pairs likely to be involved in fetal HSC migration and maintenance. Our work paves the way for research into the mechanisms underlying HSC colonization in human fetal BM and provides invaluable resources for future studies on HSC development.

WDR82-binding long noncoding RNA lncEry controls mouse erythroid differentiation and maturation.

Hematopoietic differentiation is controlled by both genetic and epigenetic regulators. Long noncoding RNAs (lncRNAs) have been demonstrated to be important for normal hematopoiesis, but their function in erythropoiesis needs to be further explored. We profiled the transcriptomes of 16 murine hematopoietic cell populations by deep RNA sequencing and identified a novel lncRNA, Gm15915, that was highly expressed in erythroid-related progenitors and erythrocytes. For this reason, we named it lncEry. We also identified a novel lncEry isoform, which was the principal transcript that has not been reported before. lncEry depletion impaired erythropoiesis, indicating the important role of the lncRNA in regulating erythroid differentiation and maturation. Mechanistically, we found that lncEry interacted with WD repeat-containing protein 82 (WDR82) to promote the transcription of Klf1 and globin genes and thus control the early and late stages of erythropoiesis, respectively. These findings identified lncEry as an important player in the transcriptional regulation of erythropoiesis.

A comprehensive RNA editome reveals that edited Azin1 partners with DDX1 to enable hematopoietic stem cell differentiation.

Adenosine-to-inosine RNA editing and the catalyzing enzyme adenosine deaminase are both essential for hematopoietic development and differentiation. However, the RNA editome during hematopoiesis and the underlying mechanisms are poorly defined. Here, we sorted 12 murine adult hematopoietic cell populations at different stages and identified 30 796 editing sites through RNA sequencing. The dynamic landscape of the RNA editome comprises stage- and group-specific and stable editing patterns, but undergoes significant changes during lineage commitment. Notably, we found that antizyme inhibitor 1 (Azin1) was highly edited in hematopoietic stem and progenitor cells (HSPCs). Azin1 editing results in an amino acid change to induce Azin1 protein (AZI) translocation to the nucleus, enhanced AZI binding affinity for DEAD box polypeptide 1 to alter the chromatin distribution of the latter, and altered expression of multiple hematopoietic regulators that ultimately promote HSPC differentiation. Our findings have delineated an essential role for Azin1 RNA editing in hematopoietic cells, and our data set is a valuable resource for studying RNA editing on a more general basis.

[PDGFRα+ Cell-Derived SCF Regulates Hematopoiesis of Adult Mice].

OBJECTIVE: To investigate the effect of PDGFRα+ stromal cells derived SCF on hematopoiesis of adult mice. METHODS: Pdgfrα-CreER; R26-tdTomato mice model was constructed, and the proportion and distribution of PDGFRα+ cells in the liver, spleen, lung, kidney and bone marrow were analyzed by flow cytometry and confocal microscopy. Then the Pdgfrα-CreER; Scf flox/flox mice model was further constructed, the Scf in PDGFRα+ was knocked out specifically, the effect of Scf-knocked out in PDGFRα+ stromal cells in the propitiation of HSPCs in the bone marrow was analyzed by flow cytometry. The effect of SCF on the proportion on number of peripheral blood cells in mice was analyzed by whole blood analyzer. RESULTS: After Scf was knocked out in PDGFRα+ stromal cells, the propitiation and number of LKS- cell, LKS+ cell, HSC, MPP1, MKP, PreGM, PreMegE, and CFU-E in the bone marrow of mice was decreased, as well as in the number of red blood cells and hemoglobin concentration of peripheral blood. However, Scf knocked out from PDGFRα+ cells showed no effect on the hematopoiesis in spleen. CONCLUSION: specific knocked out of Scf in PDGFRα+ stromal cells in adult mice can decrease the proportion of HSPCs in the bone marrow and the number of red blood cells in peripheral blood, and finally lead to anemia in mice.

Role of endoplasmic reticulum stress in disuse osteoporosis.

Osteoporosis is a major skeletal disease with low bone mineral density, which leads to an increased risk of bone fracture. Salubrinal is a synthetic chemical that inhibits dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in response to endoplasmic reticulum (ER) stress. To understand possible linkage of osteoporosis to ER stress, we employed an unloading mouse model and examined the effects of salubrinal in the pathogenesis of disuse osteoporosis. The results presented several lines of evidence that osteoclastogenesis in the development of osteoporosis was associated with ER stress, and salubrinal suppressed unloading-induced bone loss. Compared to the age-matched control, unloaded mice reduced the trabecular bone area/total area (B.Ar/T.Ar) as well as the number of osteoblasts, and they increased the osteoclasts number on the trabecular bone surface in a time-dependent way. Unloading-induced disuse osteoporosis significantly increased the expression of Bip, p-eIF2α and ATF4 in short-term within 6h of tail suspension, but time-dependent decreased in HU2d to HU14d. Furthermore, a significant correlation of ER stress with the differentiation of osteoblasts and osteoclasts was observed. Administration of salubrinal suppressed the unloading-induced decrease in bone mineral density, B.Ar/T.Ar and mature osteoclast formation. Salubrinal also increased the colony-forming unit-fibroblasts and colony-forming unit-osteoblasts. It reduced the formation of mature osteoclasts, suppressed their migration and adhesion, and increased the expression of Bip, p-eIF2α and ATF4. Electron microscopy showed that rough endoplasmic reticulum expansion and a decreased number of ribosomes on ER membrane were observed in osteoblast of unloading mice, and the abnormal ER expansion was significantly improved by salubrinal treatment. A TUNEL assay together with CCAAT/enhancer binding protein homologous protein (CHOP) expression indicated that ER stress-induced osteoblast apoptosis was rescued by salubrinal. Collectively, the results support the notion that ER stress plays a key role in the pathogenesis of disuse osteoporosis, and salubrinal attenuates unloading-induced bone loss by altering proliferation and differentiation of osteoblasts and osteoclasts via eIF2α signaling.