Expanding hematopoietic stem cell ex vivo: recent advances and technical considerations.

Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic hierarchy, which are responsible for sustaining the lifelong production of mature blood and immune cells. Due to their superior long-term regenerative capacity, HSC therapies such as stem cell transplantation have been used in a broad range of hematologic disorders. However, the rarity of this population in vivo considerably limits its clinical applications and large-scale analyses such as screening and safety studies. Therefore, ex vivo culture methods that allow long-term expansion and maintenance of functional HSCs are instrumental in overcoming the difficulties in studying HSC biology and improving HSC therapies. In this perspective, we discuss recent advances and technical considerations for three ex vivo HSC expansion methods including 1) polyvinyl alcohol-based HSC expansion, 2) mesenchymal stromal cell-HSC co-culture, and 3) two-/three-dimensional hydrogel HSC culture. This review summarizes the presentations and discussions from the 2022 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session.

CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells.

Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.

m6A RNA modifications: Key regulators of normal and malignant hematopoiesis.

Post-transcriptional RNA modifications determine RNA fate by influencing numerous processes such as translation, decay and localization. One of the most abundant RNA modifications is N6-methyladenoside (m6A), which has been shown to be important in healthy as well as malignant hematopoiesis. Several proteins representing key players in m6A RNA biology, such as m6A writers, erasers and readers, were recently reported to be essential for hematopoietic stem cell (HSC) function. In leukemia, expression of m6A regulators has been shown to be increased, opening up potential opportunities for therapeutic exploitation by targeting them in blood malignancies. These recent discoveries were the focus of the Fall 2021 International Society for Experimental Hematology New Investigators webinar. We review here the latest findings in the field of mRNA modifications in normal and malignant hematopoiesis and how this might open up novel therapeutic options.

Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data, revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes.

Alternative Polyadenylation in Stem Cell Self-Renewal and Differentiation.

Cellular function is shaped by transcriptional and post-transcriptional mechanisms, including alternative polyadenylation (APA). By directly controlling 3'- untranslated region (UTR) length and the selection of the last exon, APA regulates up to 70% of all cellular transcripts influencing RNA stability, output, and protein isoform expression. Cell-state-dependent 3'-UTR shortening has been identified as a hallmark of cellular proliferation. Hence, quiescent/dormant stem cells are characterized by long 3'-UTRs, whereas proliferative stem/progenitor cells exhibit 3'-UTR shortening. Here, the latest studies analyzing the role of APA in regulating stem cell state, self-renewal, differentiation, and metabolism are reviewed. The new role of APA in controlling stem cell fate opens novel potential therapeutic avenues in the field of regenerative medicine.

Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells.

Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin-Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135-CD48-CD150-). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset.

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1.

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.

Engineering human hematopoietic environments through ossicle and bioreactor technologies exploitation.

The bone marrow microenvironment contains cellular niches that maintain the pool of hematopoietic stem and progenitor cells and support hematopoietic maturation. Malignant hematopoietic cells also co-opt normal cellular interactions to promote their own growth and evade therapy. In vivo systems used to study human hematopoiesis have been developed through transplantation into immunodeficient mouse models. However, incomplete cross-compatibility between the murine stroma and transplanted human hematopoietic cells limits the rate of engraftment and the study of relevant interactions. To supplement in vivo xenotransplantation models, complementary strategies have recently been developed, including the use of three-dimensional human bone marrow organoids in vivo, generated from bone marrow stromal cells seeded onto osteo-inductive scaffolds, as well as the use of ex vivo bioreactor models. These topics were the focus of the Spring 2020 International Society for Experimental Hematology New Investigator webinar. We review here the latest advances in generating humanized hematopoietic organoids and how they allow for the study of novel microenvironmental interactions.

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells.

The long non-coding RNA (lncRNA) Maternally Expressed Gene 3 (Meg3) is encoded within the imprinted Dlk1-Meg3 gene locus and is only maternally expressed. Meg3 has been shown to play an important role in the regulation of cellular proliferation and functions as a tumor suppressor in numerous tissues. Meg3 is highly expressed in mouse adult hematopoietic stem cells (HSCs) and strongly down-regulated in early progenitors. To address its functional role in HSCs, we used MxCre to conditionally delete Meg3 in the adult bone marrow of Meg3mat-flox/pat-wt mice. We performed extensive in vitro and in vivo analyses of mice carrying a Meg3 deficient blood system, but neither observed impaired hematopoiesis during homeostatic conditions nor upon serial transplantation. Furthermore, we analyzed VavCre Meg3mat-flox/pat-wt mice, in which Meg3 was deleted in the embryonic hematopoietic system and unexpectedly this did neither generate any hematopoietic defects. In response to interferon-mediated stimulation, Meg3 deficient adult HSCs responded highly similar compared to controls. Taken together, we report the finding, that the highly expressed imprinted lncRNA Meg3 is dispensable for the function of HSCs during homeostasis and in response to stress mediators as well as for serial reconstitution of the blood system in vivo.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.