Self-renewing Hematopoietic Stem Cells Research Articles

Polyvinyl alcohol increased growth, migration, invasion, and sphere size in the PK-8 pancreatic ductal adenocarcinoma cell line

Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer used in eye drops, embolization particles, and artificial cartilage. It has also been shown to cause expansion of functional multipotent self-renewing hematopoietic stem cells under serum-free conditions. In this study, we examined the effects of PVA on human pancreatic ductal adenocarcinoma (PDAC) cell lines using 2-dimensional (2D) and 3D-cultures with serum-free medium. In the 2D-culture, PVA-treatment induced an aggregated colony-like appearance in PDAC cells. It increased the growth of PK-8 cells in a dose-dependent manner as well as significantly increasing migration and invasion abilities. qRT-PCR showed an increase in α2 integrin and a decrease in matrix metalloprotease levels in PVA-treated PK-8 cells. Through qRT-PCR analysis, β1 integrin expression at the mRNA level was found to be decreased; however, it was unaltered at the protein level when assessed using FACS analysis.PVA further induced mesenchymal to epithelial transition-like alterations, including increased E-cadherin and decreased Vimentin and N-cadherin expression. Four cancer stem cell (CSC) markers were higher in PVA-treated PK-8 cells compared to controls. In 3D-culture, PVA-treated PK-8 cells showed a rod-like appearance with larger sphere size and higher growth ability. qRT-PCR showed that CSC markers did not increase and 2 of 4 drug transporters had decreased in PVA-treated PK-8 cells. These findings suggest that PVA increases the growth, migration, invasion, and sphere size of PK-8 cells, but does not increase the proportion of pancreatic CSCs under 3D-culture conditions with serum-free medium.

Leptin receptor, a surface marker for a subset of highly engrafting long-term functional hematopoietic stem cells.

The hematopoietic system is sustained by a rare population of hematopoietic stem cells (HSCs), which emerge during early embryonic development and then reside in the hypoxic niche of the adult bone marrow microenvironment. Although leptin receptor (Lepr)-expressing stromal cells are well-studied as critical regulators of murine hematopoiesis, the biological implications of Lepr expression on HSCs remain largely unexplored. We hypothesized that Lepr+HSCs are functionally different from other HSCs. Using in vitro and in vivo experimental approaches, we demonstrated that Lepr further differentiates SLAM HSCs into two distinct populations; Lepr+HSCs engrafted better than Lepr-HSCs in primary transplant. Compared to Lepr-LSK cells, Lepr+LSK cells were highly enriched for extensively repopulating and self-renewing HSCs. Molecularly, Lepr+HSCs were characterized by a pro-inflammatory transcriptomic profile enriched for Type-I Interferon and Interferon-gamma (IFN-γ) response pathways, which are known to be critical for the emergence of HSCs in the embryo. We conclude that although Lepr+HSCs represent a minor subset of HSCs, they are highly engrafting cells that possess embryonic-like transcriptomic characteristics, and that Lepr can serve as a reliable marker for functional long-term HSCs, which may have potential clinical applicability.

Yap1-Scribble polarization is required for hematopoietic stem cell division and fate

AbstractYap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

BMP signaling is required for postnatal murine hematopoietic stem cell self-renewal.

Life-long production of blood from hematopoietic stem cells (HSC) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal – a cardinal feature of HSC. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSC. However, the BMP type-II receptor (BMPR-II) is highly expressed in HSC, leaving the possibility that BMP function via alternative pathways. Here, we establish that BMP signaling is required for selfrenewal of adult HSC. Through conditional knockout we show that BMPR-II deficient HSC have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMP in HSC. Indeed, a majority of primitive hematopoietic cells do not engage in SMADmediated responses downstream of BMP in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSC to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.

Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.

SummaryBone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)1,2. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined3–11. Recent advances in single cell RNA sequencing (scRNAseq) have revealed transcriptional differences amongst HSCs, providing a possible explanation for their functional heterogeneity12–17. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Differential gene expression analysis between clones with distinct behaviour unveiled an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor Tcf15 to be required, and sufficient, to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programs associated with functional stem cell heterogeneity, and identifies a mechanism for the maintenance of the self-renewing haematopoietic stem cell state.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, invivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold invivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal

SUMMARYLow protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5+ mouse long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single cell transplants and lineage tracing suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). We show that NEO1+Hoxb5+ LT-HSCs expand with age and respond to myeloablative stress in young mice while NEO1-Hoxb5+ LT-HSCs exhibit no significant change in number. Furthermore, NEO1+Hoxb5+ LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1-Hoxb5+ LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+ LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution while NEO1-Hoxb5+ LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression analysis reveals erythroid and myeloid priming in the NEO1+ fraction and association of quiescence and self-renewal-related transcription factors with NEO1- LT-HSCs. Finally, transplanted NEO1+Hoxb5+ LT-HSCs rarely generate NEO1-Hoxb5+ LT-HSCs while NEO1-Hoxb5+ LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced NEO1-Hoxb5+ LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+ LT-HSCs.

Pulsation Activates Mechanosensitive Piezo1 to Form Long-Term Hematopoietic Stem Cells

The temporal and spatial origin and development of long-term, self-renewing hematopoietic stem cells (LT-HSC) remain a mystery. The first set of definitive HSCs is born from the hemogenic endothelial cells residing in the ventral wall of the dorsal aorta (DA) of the aorta-gonad-mesonephros region during embryonic development. Blood flow- and shear-stress-mediated nitric oxide-induced vasodilation are responsible for the endothelial-to-HSC transition (EHT). However, it remains unknown why the ventral wall, and not the dorsal wall, of the DA is the restricted site of the EHT when blood flows through the entire DA and exerts shear stress on both the ventral and dorsal sides of the DA. Using single-particle tracking and fast Fourier Transform analyses of pulsating blood vessels, we demonstrate that the circumferential strain in the ventral wall, and not dorsal wall, is concurrent with and responsible for the magnitude, the site, and timing of the HSC formation. We extended our findings by developing a bioreactor to establish the functional link between pulsation in the blood vessels and HSC formation. Using serial transplant, limiting dilution, and serial replating assays, we found that pulsation mediated circumferential stretching of hemogenic endothelial cells or Piezo1 activation (Yoda1) yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using delayed-type hypersensitivity assay, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activation-derived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, & confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our CUT&RUN CHIP-Sequencing & MASSArray methylation analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. To analyze the conserved role of PIEZO1-mediated mechanosensitive mechanisms in human hematopoiesis, we employed directed differentiation of constitutive RUNX1-mCherry human induced pluripotent stem cells (iPSCs) to hemogenic endothelial cells. We found that Yoda1-mediated PIEZO1 activation stimulated human endothelial-to-hematopoietic transition. In conclusion, pulsation-mediated circumferential strain activates Piezo1 to stimulate the endothelial-to-HSC transition via the induction of Dnmt3b expression. This leads to the formation of long-term self-renewing HSCs, which can engraft and reconstitute to multi-lineage, adult blood upon serial transplantations. Our identification of a novel biomechanical cue unravels the physiological mystery in HSC formation in the ventral wall of the DA. We also establish its cross-talk with mechanosensitive and epigenetic mechanisms to produce functional, long-term HSCs that reconstitute to form normal adult blood. This yields the therapeutic promise of developing transgene-free LT-HSC-based cellular therapies for the treatment of human blood disorders. Disclosures No relevant conflicts of interest to declare.

Single Cell Profiling of DNMT3A-Mutant Progenitors Reveals LY86 As a Novel Pre-Leukemia Marker and Potential Therapeutic Target

Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that initiated and sustained by self-renewing hematopoietic stem and progenitor cells (HSPC). Mutations in the de novo DNA methyltransferase 3A (DNMT3A) gene occur in approximately 25% of adult acute myeloid leukemias (AML). Although the mechanisms through which such mutations promote leukemogenesis remain unclear, we have previously shown that loss of the DNMT3A can inhibit normal hematopoietic differentiation (Challen, Nature Genetics, 2011), accounting for the emergence of DNMT3A-HSC clones as a predisposition to hematological malignancies (Yang, Cancer Cell, 2015). Therapies that selectively eliminate the initiating pre-leukemic population would greatly improve outcomes for affected patients. However, the identification as well as selective elimination of such a distinct population has been problematic because of the considerable overlap in gene expression profiles with bulk normal hematopoietic stem cells. Molecular targets during leukemia development have not been well elucidated due to lack of the real definitive markers, which is a significant knowledge gap and barrier for understanding clonal leukemogenesis and therapeutic applications. Single-cell RNA sequencing has emerged as a powerful tool to analyze new cell types, cellular heterogeneity and cell differentiation routes. This technique made important contributions to our understanding of hematopoietic stem and cancer cell heterogeneity and selective resistance of cancer cell subpopulations to molecularly targeted cancer therapies. To identify early events involved in pre-leukemic transformation, we have performed single-cell RNA-sequencing (scRNA-seq) in WT and Dnmt3a KO mice. Flow cytometry sorted wild-type and pre-leukemic Dnmt3a KO HSPC cells were captured using 10X genomics chromium platform. After genome mapping, dimensional reduction, and clustering using Cell ranger pipeline, we generated transcriptome data and integrated the data sets using Seurat. Approximately 8,000 cells from each group were sequenced, and each cell expressed 1800-4500 genes. Graph-based clustering analysis revealed 16 unique cell clusters in both WT and DNMT3A KO mice. Interestingly, when compared with WT mice, we observed a 10-fold expansion of a single cell cluster in Dnmt3a KO cells before the advent of overt leukemia. This cluster co-expresses several stem cell genes including well-known leukemic stem cell surface markers such as CD47, as well as several novel genes. Some of these novel genes, encode cell surface proteins such as Ly6c2 and Ly86. We further validated protein expressions in AML cell lines and primary AML blast. In conclusion, the discovery of novel cluster in DNMT3A KO mice, and the relative abundance of this cluster in pre-leukemic stage of DNMT3A KO mice indicates that they promote leukemogenesis and offers an opportunity to specifically target DNMT3A mutant pre-leukemic cells using T cell immunotherapy. Disclosures No relevant conflicts of interest to declare.

Fli-1 Transcriptionally Integrates Microenvironmental Cues Sensing By Self-Renewing Hematopoietic Stem and Progenitor Cells

During adulthood and embryogenesis, fate decisions of hematopoietic stem and progenitor cells (HSPCs), such as specification, self-renewal, and differentiation are tightly regulated by their neighboring niche cells. Moreover, distinct types of niches supply differential cues to direct alternative cell fates for HSPCs. Yet, currently the intrinsic mechanisms balancing HSPC response obliqueness to microenvironmental signals are unknown. Friend Leukemia integration-1 (Fli-1), is an ETS transcription factor expressed by vascular beds and hematopoietic lineages. Fli-1 belongs to the "heptad factors" which are hypothesized to specify and sustain a hematopoietic cell fate. While Fli-1 overexpression is linked to leukemia, the functional role Fli-1 plays in HSPC specification and maintenance remains undefined. We show that inducible deletion of Fli-1 using a Rosa-CreERT2 transgenic adult mice (Fli-1ROSAΔ), results in a rapid thrombocytopenia-associated mortality. Transplantation of Fli-1ROSAΔ bone marrow (BM) cells into WT recipients, to exclude vascular-mediated defects, followed by induction of Fli-1 deletion, resulted with the same phenotype. In a set of modulated competitive transplantation experiments (differential induction time points pre- or post-transplant), we observed defective ability of Fli-1ROSAΔ HSPCs to lodge, engraft, and to sustain hematopoiesis post repopulation. Fli-1 deficient HSPCs exhibited reduced quiescent cell cycling status, a hallmark of stemness, and displayed enhanced apoptosis. Thus, Fli-1 is essential for previously unrecognized cell-autonomous HSPC functions. To determine whether Fli-1 modulates HSPC specification, Fli-1 was conditionally deleted using a developmental VE-cadherin (CDH5)-Cre transgenic model (Fli-1CDH5Δ). This resulted with premature mortality of Fli-1CDH5Δ embryos, accompanied with a hemorrhagic phenotype. Reduced numbers of hematopoietic cells were still detected in the AGM of e10.5 Fli-1CDH5Δ embryos. Conditional Fli-1 deletion using a developmental hematopoietic Vav-1 Cre transgenic model (Fli-1Vav-1Δ) resulted again with premature mortality. Reduced presence of embryonic Fli-1Vav-1Δ liver HSPCs was observed at e12.5. We also applied two in vitro co-culture systems, to study Fli-1 in endothelial to hematopoietic transition (EHT). First, isolated hemogenic endothelial cells (HEC) from WT and Fli-1ROSAΔ embryos were co-cultured with AGM-derived vascular niche. HECs isolated from Fli-1ROSAΔ AGM were still able to convert to CD45+ cells, however these cells did not expand on a vascular niche. Secondly, we have applied an endothelial to hematopoietic reprogramming system in which isolated lung ECs are virally introduced with DOX inducible FosB, Gfi1, Runx1, and Spi1 (FGRS) factors and co-cultured with vascular niche cells. Both WT and Fli-1ROSAΔ ECs were able to acquire a hemogenic like state resulting with a final capacity to convert into hematopoietic cells. Again, Fli-1ROSAΔ cells displayed lesser numbers of CD45+ cells at the end point, presumably due to impaired interaction with the vascular niche. Indeed, reduced expansion capacity was observed both for mature CD45+ and for HSPC derived from Fli-1CDH5Δ AGM region. Adult Fli-1ROSAΔ HSPCs exhibited the same niche-dependent expansion defect. Induction of Fli-1 deletion in vitro in adult HSPCs revealed loss of dependency on vascular niche inductive signals, as no additive expansion effect was observed for Fli-1ROSAΔ HSPCs in the presence of a vascular niche. Hence, Fli-1 is essential for HSPC expansion rather than hematopoietic specification. Differential RNA-seq analysis combined with epigenetic studies of expanding WT and Fli-1ROSAΔ HSPCs, revealed dysregulation of Fli-1-controlled pathways involved in transduction of microenvironmental signals for self-renewal. Unexpectedly, H3K27Ac analysis, a marker for transcriptional priming, revealed increased global acetylation of Fli-1ROSAΔ HSPCs' chromatin. Therefor, Fli-1 may not only perform as transcription activator, but foremostly as a genomic suppressor via modulation of histone acetylation status. Decrypting the mechanism(s) by which Fli-1 orchestrates HSPC self-renewal, may promote an improved expansion protocol of human HSPC pre-transplantation, and provide additional insights for microenvironmental sensing by Fli-1-dependent leukemic cells. Disclosures No relevant conflicts of interest to declare.

Inflammageing of Hematopoietic Stem Cells Is Driven By IL-1

Introduction: Lifelong blood production is sustained through a stepwise differentiation program by a limited number of self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). Hematopoietic cell development is tightly controlled by both cell-intrinsic and -extrinsic factors and its dysregulation can lead to aplasia or neoplasia. During ageing, HSCs increase in number, reduce self-renewal capacity on a per cell basis, skew towards myeloid differentiation, and show less efficient bone marrow (BM)-homing ability. We here evaluated how and to what extend HSC-extrinsic factors determine HSC behaviour during aging. Methods, Results and Discussion: To screen for aging-associated extrinsic factors, we performed antibody based protein arrays and transcriptome analysis with total BM of young (6-8 week old) versus aged (2 year old) animals. This demonstrated that RANTES, MIP-2, IL-1α and IL-1β are significantly upregulated in aged BM at both the protein as well as the RNA level. ELISA of peripheral blood (PB) serum and BM lysates indicated that IL-1α and IL-1β are locally increased and produced in BM, but are not significantly increased in PB serum. Further, qPCR of various BM cell types of hematopoietic (myeloid, lymphoid and progenitor cells) and non-hematopoietic/stromal origin indicated that multiple cell types upregulate Il1α and Il1β, with highest increase being derived from myeloid hematopoietic cells. This raised the possibility that elevated IL-1 is a result of an inflammatory response to circulating pathogen-derived compounds, possibly of bacterial origin. Indeed, we previously demonstrated that steady-state levels of granulopoiesis in young steady-state mice depend on heat-resistant microbiota-derived compounds (M.Balmer et al., The Journal of Immunology 2014). To prospectively test the role of IL-1-induced signalling and the microbiome during aging, we investigated the ageing-associated phenotype of HSCs in young and aged IL1RIKO mice and in young and aged germ-free mice. Both IL1RIKO and germ-free aged mice had lower counts of platelets and neutrophils in PB, and lower frequency of LT-HSCs (LKS Flt3-CD34-CD48-CD150+) in BM, compared to aged WT mice. Moreover, aged IL1RIKO LT-HSCs showed improved lymphoid lineage repopulation upon transplantation into lethally irradiated WT mice, compared to LT-HSCs of aged WT mice that demonstrated the known myeloid-biased lineage output. Interestingly, LT-HSCs from aged germ-free mice also demonstrated lymphoid-biased lineage differentiation as observed from young mice. In line with this finding, no difference was observed in IL-1α and IL-1β protein concentrations in BM lysates from young and aged germ-free mice. To test if IL-1 increase in aged steady-state mouse BM is indeed dependent on ligation of pattern recognition receptors and consecutive signalling, we analysed MyD88 and Trif KO mice, respectively. Both aged KO mice showed compared to WT mice reduced BM IL-1 levels and a reduced ageing-phenotype of HSCs, with the most profound difference in Trif KO mice. Interestingly, this correlates with our previous finding on pathogen-Induced TLR4-TRIF innate immune signaling in HSCs, inducing reduced competitive fitness (Takizawa et al., Cell Stem Cell 2017). Conclusions: Our data demonstrate that ageing associated phenotype and myeloid-biased differentiation of HSCs is a result of signals derived from the microbiome, that act through increased IL-1 signalling, locally in BM. Disclosures No relevant conflicts of interest to declare.

Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia.

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34+ cells, which showed marked proliferative advantage and phenotypic correction both invitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.

Interleukin-12 supports in vitro self-renewal of long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) self-renew or differentiate through division. Cytokines are essential for inducing HSC division, but the optimal cytokine combination to control self-renewal of HSC in vitro remains unclear. In this study, we compared the effects of interleukin-12 (IL-12) and thrombopoietin (TPO) in combination with stem cell factor (SCF) on in vitro self-renewal of HSCs. Single-cell assays were used to overcome the heterogeneity issue of HSCs, and serum-free conditions were newly established to permit reproduction of data. In single-cell cultures, CD150+CD48−CD41−CD34−c-Kit+Sca-1+lineage− HSCs divided significantly more slowly in the presence of SCF+IL-12 compared with cells in the presence of SCF+TPO. Serial transplantation of cells from bulk and clonal cultures revealed that TPO was more effective than IL-12 at supporting in vitro self-renewal of short-term (<6 months) HSCs, resulting in a monophasic reconstitution wave formation, whereas IL-12 was more effective than TPO at supporting the in vitro self-renewal of long-term (>6 months) HSCs, resulting in a biphasic reconstitution wave formation. The control of division rate in HSCs appeared to be crucial for preventing the loss of self-renewal potential from their in vitro culture.

1028 - MITOCHONDRIA METABOLISM IN QUIESCENT AND CYCLING HEMATOPOIETIC STEM CELLS

Cellular metabolism is an area of recent intense research interest. However, the metabolic requirements for adaptations of stem cells to their niches remain largely unaddressed. The cell metabolism may be quite different in quiescent and cycling HSCs. Most of the hematopoietic stem cells (HSCs) within the bone marrow show quiescent state. By contrast, upon stress hematopoiesis, HSCs actively divide to regenerate hematopoietic system with the appropriate balance between self-renewal and differentiation divisions. Here we have compared mitochondrial metabolism between the quiescent state and cycling state in various conditions. First, we will show that suppression of Ca2+ influx and subsequent mitochondrial membrane potential contributes to the maintenance of HSCs by slowing cell divisions. Next, under the stress hematopoiesis such as thrombopoietin (Thpo) administration or oppositely, in Thpo-deficient mice, cell metabolism is drastically changed accompanying by the shift of the HSC subpopulation. Finally, using ATG7-deficient mice, we want to suggest that autophagy process is involved in the maintaining quiescence in adult HSCs, but not in neonatal HSCs, which show different nature in cell metabolism from that of adult quiescent HSCs. We will clarify the common and different points of cycling neonatal and dormant adult HSCs, as well as Thpo-deficient HSCs. Then, we would like to propose two types (dormant and cycling) of self-renewing HSCs.

MEGAKARYOCYTE-LINEAGE BIAS AND RESTRICTION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS

Hematopoietic stem cells (HSCs), defined by long-term self-renewal and ability to replenish all blood lineages, are molecularly and functionally heterogeneous. This heterogeneity might have important implications in both steady-state and emergency hematopoiesis, as well as aging and hematological malignancies. We previously uncovered a highly organized framework of lineage-biased and lineage-restricted HSC fates through sensitive tracking of post-transplantation contribution of hundreds of single HSCs from adult murine bone marrow (BM) (Carrelha et al. Nature 2018). Most notably, we identified HSCs that in vivo contribute stably, effectively, and exclusively towards the megakaryocyte (Mk) branch of the hematopoietic tree, despite possessing multipotency. No long-term self-renewing HSCs contribute exclusively to any other lineage upon transplantation. We also identified Mk/erythroid/myeloid and Mk/erythroid/myeloid/B-cell restricted outputs. Contrary to previous studies, we found no evidence for long-term adult BM HSCs with sustained lymphoid bias. Recently we also performed single-cell transplantations of HSCs from late gestation mouse embryos (E19.5). We found that these fetal liver and fetal BM HSCs undergo almost exclusively multilineage fates, rarely producing lineage-restricted outputs, which suggests that the lineage biases and restrictions of adult HSCs are established in the BM postnatally. Through fate-mapping analysis with an inducible Vwf-Cre reporter we demonstrated that Mk-biased HSCs play an important role in unperturbed adult BM hematopoiesis. We have further observed that Mk-biased HSCs play a more prominent role in the rapid replenishment of platelets following an acute insult with 5-fluorouracil. In agreement with this data, single transplanted HSCs with Mk-restricted fate replenish platelets more rapidly than HSCs undergoing a multilineage fate.

Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

UM171 Induces a Homeostatic Inflammatory Response Supporting Human HSC Self-Renewal

Elucidation of the molecular cues required to balance adult stem cell self-renewal and differentiation is critical for advancing cellular therapies. Herein, we report that the hematopoietic stem cell (HSC) self-renewal agonist UM171 triggers a balanced pro- and anti-inflammatory/detoxification network that relies on NFKB-CD86 activation and protein C receptor-dependent ROS detoxification, respectively. We demonstrate that within this network, EPCR serves as a critical protective component as its deletion hypersensitizes primitive hematopoietic cells to pro-inflammatory signals resulting in compromised stem cell function. Conversely, abrogation of the pro-inflammatory activity of UM171 through either CD86 suppression, treatment with dexamethasone, cAMP elevating agents or NFKB inhibitors abolishes EPCR upregulation and HSC expansion. Together, these results show that UM171 stimulates ex vivo HSC expansion by establishing a critical balance between key pro- and anti-inflammatory mediators of self-renewal.

Mitochondrial Role in Stemness and Differentiation of Hematopoietic Stem Cells

Quiescent and self-renewing hematopoietic stem cells (HSCs) rely on glycolysis rather than on mitochondrial oxidative phosphorylation (OxPHOS) for energy production. HSC reliance on glycolysis is considered an adaptation to the hypoxic environment of the bone marrow (BM) and reflects the low energetic demands of HSCs. Metabolic rewiring from glycolysis to mitochondrial-based energy generation accompanies HSC differentiation and lineage commitment. Recent evidence, however, highlights that alterations in mitochondrial metabolism and activity are not simply passive consequences but active drivers of HSC fate decisions. Modulation of mitochondrial activity and metabolism is therefore critical for maintaining the self-renewal potential of primitive HSCs and might be beneficial for ex vivo expansion of transplantable HSCs. In this review, we emphasize recent advances in the emerging role of mitochondria in hematopoiesis, cellular reprograming, and HSC fate decisions.