Restricted Progenitors Research Articles

Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Defining the contribution of Troy-positive progenitor cells to the mouse esophageal epithelium

Progenitor cells adapt their behavior in response to tissue demands. However, the molecular mechanisms controlling esophageal progenitor decisions remain largely unknown. Here, we demonstrate the presence of a Troy (Tnfrsf19)-expressing progenitor subpopulation localized to defined regions along the mouse esophageal axis. Lineage tracing and mathematical modeling demonstrate that Troy-positive progenitor cells are prone to undergoing symmetrical fate choices and contribute to esophageal tissue homeostasis long term. Functionally, TROY inhibits progenitor proliferation and enables commitment to differentiation without affecting fate symmetry. Whereas Troy expression is stable during esophageal homeostasis, progenitor cells downregulate Troy in response to tissue stress, enabling proliferative expansion of basal cells refractory to differentiation and reestablishment of tissue homeostasis. Our results demonstrate functional, spatially restricted progenitor heterogeneity in the esophageal epithelium and identify how dynamic regulation of Troy coordinates tissue generation.

Differentiation route determines the functional outputs of adult megakaryopoiesis

Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.

The Adult Carotid Body: A Germinal Niche at the Service of Physiology.

The carotid body is the most relevant oxygen sensor in mammalian organisms. This organ helps to detect acute changes in PO2, but it is also crucial for the organismal adaptation to a maintained hypoxemia. Profound angiogenic and neurogenic processes take place in the carotid body to facilitate this adaptation process. We have described a plethora of multipotent stem cells and restricted progenitors, from both vascular and neuronal lineages, existing in the quiescent normoxic carotid body, ready to contribute to organ growth and adaptation upon the arrival of the hypoxic stimulus. Our deep understanding of the functioning of this stunning germinal niche will very likely facilitate the management and treatment of an important group of diseases that course with carotid body over-activation and malfunction.

Low-intensity illumination for lensless digital holographic microscopy with minimized sample interaction.

Exposure to laser light alters cell culture examination via optical microscopic imaging techniques based on label-free coherent digital holography. To mitigate this detrimental feature, researchers tend to use a broader spectrum and lower intensity of illumination, which can decrease the quality of holographic imaging due to lower resolution and higher noise. We study the lensless digital holographic microscopy (LDHM) ability to operate in the low photon budget (LPB) regime to enable imaging of unimpaired live cells with minimized sample interaction. Low-cost off-the-shelf components are used, promoting the usability of such a straightforward approach. We show that recording data in the LPB regime (down to 7 µW of illumination power) does not limit the contrast or resolution of the hologram phase and amplitude reconstruction compared to regular illumination. The LPB generates hardware camera shot noise, however, to be effectively minimized via numerical denoising. The ability to obtain high-quality, high-resolution optical complex field reconstruction was confirmed using the USAF 1951 amplitude sample, phase resolution test target, and finally, live glial restricted progenitor cells (as a challenging strongly absorbing and scattering biomedical sample). The proposed approach based on severely limiting the photon budget in lensless holographic microscopy method can open new avenues in high-throughout (optimal resolution, large field-of-view, and high signal-to-noise-ratio single-hologram reconstruction) cell culture imaging with minimized sample interaction.

Human induced pluripotent stem cell engineering establishes a humanized mouse platform for pediatric low-grade glioma modeling

A major obstacle to identifying improved treatments for pediatric low-grade brain tumors (gliomas) is the inability to reproducibly generate human xenografts. To surmount this barrier, we leveraged human induced pluripotent stem cell (hiPSC) engineering to generate low-grade gliomas (LGGs) harboring the two most common pediatric pilocytic astrocytoma-associated molecular alterations, NF1 loss and KIAA1549:BRAF fusion. Herein, we identified that hiPSC-derived neuroglial progenitor populations (neural progenitors, glial restricted progenitors and oligodendrocyte progenitors), but not terminally differentiated astrocytes, give rise to tumors retaining LGG histologic features for at least 6 months in vivo. Additionally, we demonstrated that hiPSC-LGG xenograft formation requires the absence of CD4 T cell-mediated induction of astrocytic Cxcl10 expression. Genetic Cxcl10 ablation is both necessary and sufficient for human LGG xenograft development, which additionally enables the successful long-term growth of patient-derived pediatric LGGs in vivo. Lastly, MEK inhibitor (PD0325901) treatment increased hiPSC-LGG cell apoptosis and reduced proliferation both in vitro and in vivo. Collectively, this study establishes a tractable experimental humanized platform to elucidate the pathogenesis of and potential therapeutic opportunities for childhood brain tumors.

Dbx1 controls the development of astrocytes of the intermediate spinal cord by modulating Notch signaling.

Significant progress has been made in elucidating the basic principles that govern neuronal specification in the developing central nervous system. In contrast, much less is known about the origin of astrocytic diversity. Here, we demonstrate that a restricted pool of progenitors in the mouse spinal cord, expressing the transcription factor Dbx1, produces a subset of astrocytes, in addition to interneurons. Ventral p0-derived astrocytes (vA0 cells) exclusively populate intermediate regions of spinal cord with extraordinary precision. The postnatal vA0 population comprises gray matter protoplasmic and white matter fibrous astrocytes and a group of cells with strict radial morphology contacting the pia. We identified that vA0 cells in the lateral funiculus are distinguished by the expression of reelin and Kcnmb4. We show that Dbx1 mutants have an increased number of vA0 cells at the expense of p0-derived interneurons. Manipulation of the Notch pathway, together with the alteration in their ligands seen in Dbx1 knockouts, suggest that Dbx1 controls neuron-glial balance by modulating Notch-dependent cell interactions. In summary, this study highlights that restricted progenitors in the dorsal-ventral neural tube produce region-specific astrocytic subgroups and that progenitor transcriptional programs highly influence glial fate and are instrumental in creating astrocyte diversity.

Post natal expression of Prx1 labels appendicular restricted progenitor cell populations of multiple tissues.

Currently, there is no consensus whether there is a single or multiple postnatal stem cell population(s) that contribute to skeletal homeostasis and postnatal bone formation. A known population of cells that express Prx1 contributes to postnatal bone formation. Prx1 expression also connotes calvaria and appendicular tissues during embryonic development. A transgenic tamoxifen inducible Prx1 reporter mouse was used for lineage tracking, to characterize the postnatal contribution of Prx1 expressing cells in skeletal homeostasis and bone formation. Under homeostatic conditions Prx1 labeling gave rise to a transient yet rapid turnover cell population at the periosteal and endosteal surfaces, along muscle fibers, and within the medial layers of vessels both within the muscle and marrow compartments of the appendicular skeleton. Fracture and ectopic bone formation of both fore and hind limbs showed recruitment and expansion of Prx1-derived cells in newly formed bone tissues. Prx1 labeled cells were limited or absent at axial skeletal sites during both homeostasis and after induction of bone formation. Last, Prx1-derived cells differentiated into multiple cell lineages including vascular smooth muscle, adipose, cartilage, and bone cells. These results show that Prx1 expression retained its embryonic tissue specification and connotes a stem/progenitor cell populations of mesenchymal tissue progenitors.

Metabolic regulation of somatic stem cells in vivo.

Metabolism has been studied mainly in cultured cells or at the level of whole tissues or whole organisms in vivo. Consequently, our understanding of metabolic heterogeneity among cells within tissues is limited, particularly when it comes to rare cells with biologically distinct properties, such as stem cells. Stem cell function, tissue regeneration and cancer suppression are all metabolically regulated, although it is not yet clear whether there are metabolic mechanisms unique to stem cells that regulate their activity and function. Recent work has, however, provided evidence that stem cells do have a metabolic signature that is distinct from that of restricted progenitors and that metabolic changes influence tissue homeostasis and regeneration. Stem cell maintenance throughout life in many tissues depends upon minimizing anabolic pathway activation and cell division. Consequently, stem cell activation by tissue injury is associated with changes in mitochondrial function, lysosome activity and lipid metabolism, potentially at the cost of eroding self-renewal potential. Stem cell metabolism is also regulated by the environment: stem cells metabolically interact with other cells in their niches and are able to sense and adapt to dietary changes. The accelerating understanding of stem cell metabolism is revealing new aspects of tissue homeostasis with the potential to promote tissue regeneration and cancer suppression.

3203 – IDENTIFICATION OF DIFFERENTIATION ROOTS OF HEMATOPOIETIC STEM CELLS BY A PAIRED-DAUGHTER ASSAY COMBINED WITH MULTIPLE BARCODING

Hematopoietic stem cells (HSCs) produce all blood cells with self-renewal potential. HSCs maintain their compartments by symmetric and asymmetric cell division. A paired daughter cell (PDC) assay combined with single-cell transplantation is a powerful method to compare lineage outputs of two HSC-daughter cells. However, it is difficult to analyze the large number of pairs at once. Here, we have developed a PDC assay combined with DNA barcoding to overcome the limitation. Briefly, we labeled HSCs by lentiviral DNA barcode in vitro, split them in half when most of the HSCs divide once, which can split HSC-daughter cells with fifty-fifty chance, transplanted each half to each lethally irradiated recipient mouse and analyzed barcodes detected as pairs later. This method enabled us to analyze the large number of paired-daughter cells of HSCs only from two recipient mice. The most common HSC-division pattern was balanced HSC (bHSC)-bHSC. We also detected the other following division patterns with frequencies in the following order: lymphoid-biased HSC (lyHSC)-lymphoid restricted progenitor (LyRP), myeloid-biased HSC (myHSC)-LyRP, short-term HSC (ST-HSC)-ST-HSC, myHSC-myHSC and myHSC-bHSC. Most of the HSCs except lyHSCs were generated by symmetric division. Differentiated progenitor outputs were often detected as a paired-daughter cell of lyHSC or myHSC, not bHSC, suggesting that differentiation of HSCs begins with lyHSCs and myHSCs and bHSCs primarily maintain the stem cell pool. Single-cell RNA-sequencing analysis and secondary transplantation confirmed the current HSC hierarchy in which myHSCs are at the apex, producing bHSCs and lyHSCs. Interestingly, a few myHSCs behaved as latent HSCs which don't produce any progenies. In summary, the combination of the PDC assay and DNA barcoding revealed the distinct division pattern and differentiation capacity in each HSC subtype. Hematopoietic stem cells (HSCs) produce all blood cells with self-renewal potential. HSCs maintain their compartments by symmetric and asymmetric cell division. A paired daughter cell (PDC) assay combined with single-cell transplantation is a powerful method to compare lineage outputs of two HSC-daughter cells. However, it is difficult to analyze the large number of pairs at once. Here, we have developed a PDC assay combined with DNA barcoding to overcome the limitation. Briefly, we labeled HSCs by lentiviral DNA barcode in vitro, split them in half when most of the HSCs divide once, which can split HSC-daughter cells with fifty-fifty chance, transplanted each half to each lethally irradiated recipient mouse and analyzed barcodes detected as pairs later. This method enabled us to analyze the large number of paired-daughter cells of HSCs only from two recipient mice. The most common HSC-division pattern was balanced HSC (bHSC)-bHSC. We also detected the other following division patterns with frequencies in the following order: lymphoid-biased HSC (lyHSC)-lymphoid restricted progenitor (LyRP), myeloid-biased HSC (myHSC)-LyRP, short-term HSC (ST-HSC)-ST-HSC, myHSC-myHSC and myHSC-bHSC. Most of the HSCs except lyHSCs were generated by symmetric division. Differentiated progenitor outputs were often detected as a paired-daughter cell of lyHSC or myHSC, not bHSC, suggesting that differentiation of HSCs begins with lyHSCs and myHSCs and bHSCs primarily maintain the stem cell pool. Single-cell RNA-sequencing analysis and secondary transplantation confirmed the current HSC hierarchy in which myHSCs are at the apex, producing bHSCs and lyHSCs. Interestingly, a few myHSCs behaved as latent HSCs which don't produce any progenies. In summary, the combination of the PDC assay and DNA barcoding revealed the distinct division pattern and differentiation capacity in each HSC subtype.

CEBPA Directly Binds Ribosomal DNA and Promotes Ribosomal RNA Transcription in Myeloid Progenitors

Hematopoietic stem cells (HSCs) form a hierarchy of lineage restricted progenitor cells to produce mature hematopoietic cells that vary in function, size, proliferation, and protein synthesis rates. Different hematopoietic cells also vary in the rate of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been identified by their prominent nucleoli, indicating high ribosome biogenesis rates (Fig A). Ribosome biogenesis is an extremely energy intensive process begins with transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA) which further processed into the generation of mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig B). This process is highly dynamic and regulated at the level of rRNA transcription. Despite cell-type and disease-specific variations, rRNA transcription has long been considered a housekeeping process. Hence, cell or tissue type-specific regulation of rRNA transcription has rarely been explored.To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we mapped 2200 publicly available ChIP-Seq datasets representing 249 hematopoietic transcription factors (TFs) and epigenetic factors to create an atlas of hematopoietic TF-rDNA binding. We identified CEBPA that shows consistent and abundant binding to rDNA at a conserved, previously unknown motif in both species (Fig C). CEBPA is a myeloid lineage specific TF whose knockout leads to complete loss of all myeloid lineage cells. It is also frequently mutated (10%) in AML patients. So we picked CEBPA to further characterize its role in rRNA transcription.Since CEBPA deletion causes loss of granulocyte-monocyte progenitors (GMPs), we used the mouse HoxA9-ER cell line (which closely resembles GMPs). To study the immediate consequences of CEBPA loss, We generated a stable degron cell line by biallelically fusing FKBP degron into endogenous loci of Cebpa, enabling to rapidly degrade endogenous CEBPA protein on treatment with dTagV ligand (Fig D, E). To precisely quantify the rate of rRNA transcription, we developed a novel assay called ‘47S-FISH-Flow’ that involves hybridizing fluorescent oligos unique to 5' end of 47S pre-rRNA, which only marks newly synthesized nascent rRNA in the nucleolus, and quantify using flow cytometry (Fig F, G). We found that depleting CEBPA caused rapid decrease in 47S rRNA level and occupancy of Pol I on rDNA (Fig H, I).In summary, we found that myeloid lineage specific TF CEBPA abundantly binds to a conserved motif in rDNA and the depletion of CEBPA rapidly reduces nascent 47S rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Tracking By Time Lapse Imaging Confirms Thrombopoietin Promotes Megakaryocytic-Erythroid Progenitor Self Renewal, but Does Not Instruct Lineage Commitment

The molecular mechanisms underlying lineage commitment of stem and progenitor cells have implications for deriving specific cell types in vitro for regenerative medicine purposes. Current approaches to derive transfusable amounts of erythrocytes and platelets fall short of producing physiologically relevant amounts of each cell type. Thrombopoietin (TPO), commonly used in culture systems to increase platelet yield from cultured megakaryocytes, increases megakaryocyte (Mk) numbers and promotes platelet production. With the goal of generating adequate quantities of transfusion products in mind, establishing differentiation cultures that start with highly purified progenitors with Mk and E lineage potential as well as expansion potential is a very attractive option. However, a pervasive misconception exists that supplementing MEP cultures with TPO will increase the frequency with which the MEP produce megakaryocyte-committed daughter cells, thereby increasing the pool of lineage-restricted progenitor cells with high expansion capacity that ultimately yield higher numbers of transfusable platelets. We investigated whether TPO plays a role in instructing Megakaryocytic-Erythroid Progenitor (MEP) [1] commitment to the Mk lineage utilizing our previously presented time-lapse imaged colony forming unit assay and cell tracking approach [2]. This allowed us to calculate the frequency with which MEP give rise to lineage restricted progenitors in control conditions (with SCF, IL3, IL6, EPO, and TPO) compared to conditions lacking TPO to test the hypothesis that absence of TPO may diminish the frequency with which MEP give rise to Mk-restricted progenitors.We found that in the absence of TPO, MEP colony forming efficiency significantly (p<0.05) dropped by 22.4%. Importantly, the distribution of colony types was unaffected by the absence of TPO (Figure 1). By time-lapse imaging we observed the frequency with which MEP commit to the E lineage versus the Mk lineage to be approximately 2:1 in both control and -TPO conditions, demonstrating that TPO does not affect MEP lineage commitmentTime-lapse imaging also confirmed that MEP cultured without TPO underwent significantly (p<0.05) fewer symmetric self-renewal divisions resulting in less expansion of MEP, as well as less asymmetric self-renewal divisions resulting in more rapid exhaustion of MEP (Figure 2). 80% of control MEP divisions resulted in self renewal for an average of 8 generations and expansion divisions dropped below 10% after the 6 th generation, whereas MEP cultured without TPO lost the same propensity to self-renew within 3 generations. This confirms that TPO supports MEP self-renewal and expansion, similarly to how TPO supports HSC expansion [3].Cell death rates were at least 5-fold higher in the absence of TPO compared to control culture conditions containing TPO, and this effect was observed in both E and Mk-destined cells, demonstrating that TPO supports survival of both E and Mk-destined progenitors (p<0.05; Figure 3). Of note, CD41 mean fluorescence intensity was 2-fold dimmer in Mk progeny in the -TPO condition compared to control condition after 6 days in culture, which could indicate a delay in megakaryocytic maturation resulting in slower up-regulation of CD41.Our results confirm the hypothesis that TPO allows for greater self-renewal of MEP, as well as the survival of both the E and Mk-destined progenitors, however TPO is not instructive in MEP lineage commitment as evidenced by no change in the frequency of colony types. These findings also support the use of TPO in cultures of less committed progenitors to promote expansion leading to higher production of transfusable cell types. Further uses of this approach can include measuring kinetics of cellular processes such as division rate and motility, which may correlate with cell state and permit predictions of the probability of lineage choices downstream of specific progenitor populations.

Assessment of Immunological Potential of Glial Restricted Progenitor Graft In Vivo-Is Immunosuppression Mandatory?

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, causing motor neuron and skeletal muscle loss and death. One of the promising therapeutic approaches is stem cell graft application into the brain; however, an immune reaction against it creates serious limitations. This study aimed to research the efficiency of glial restricted progenitors (GRPs) grafted into murine CNS (central nervous system) in healthy models and the SOD1G93A ALS disease model. The cellular grafts were administered in semiallogenic and allogeneic settings. To investigate the models of immune reaction against grafted GRPs, we applied three immunosuppressive/immunomodulatory regimens: preimplantation factor (PiF); Tacrolimus; and CTLA-4, MR1 co-stimulatory blockade. We tracked the cells with bioluminescence imaging (BLI) in vivo to study their survival. The immune response character was evaluated with brain tissue assays and multiplex ELISA in serum and cerebrospinal fluid (CSF). The application of immunosuppressive drugs is disputable when considering cellular transplants into the immune-privileged site/brain. However, our data revealed that semiallogenic GRP graft might survive inside murine CNS without the necessity to apply any immunomodulation or immunosuppression, whereas, in the situation of allogeneic mouse setting, the combination of CTLA-4, MR1 blockade can be considered as the best immunosuppressive option.

Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states

Notch signaling is required to repress the formation of vertebrate cone photoreceptors and to maintain the proliferative potential of multipotent retinal progenitor cells. However, the mechanism by which Notch signaling controls these processes is unknown. Recently, restricted retinal progenitor cells with limited proliferation capacity and that preferentially generate cone photoreceptors have been identified. Thus, there are several potential steps during cone genesis that Notch signaling could act. Here we use cell type specific cis-regulatory elements to localize the primary role of Notch signaling in cone genesis to the formation of restricted retinal progenitor cells from multipotent retinal progenitor cells. Localized inhibition of Notch signaling in restricted progenitor cells does not alter the number of cones derived from these cells. Cell cycle promotion is not a primary effect of Notch signaling but an indirect effect on progenitor cell state transitions that leads to depletion of the multipotent progenitor cell population. Taken together, this suggests that the role of Notch signaling in cone photoreceptor formation and proliferation are both mediated by a localized function of Notch in multipotent retinal progenitor cells to repress the formation of restricted progenitor cells.

Niches that regulate stem cells and hematopoiesis in adult bone marrow.

In mammals, hematopoietic stem cells (HSCs) engage in hematopoiesis throughout adult life within the bone marrow, where they produce the mature cells necessary to maintain blood cell counts and immune function. In the bone marrow and spleen, HSCs are sustained in perivascular niches (microenvironments) associated with sinusoidal blood vessels-specialized veins found only in hematopoietic tissues. Endothelial cells and perivascular leptin receptor+ stromal cells produce the known factors required to maintain HSCs and many restricted progenitors in the bone marrow. Various other cells synthesize factors that maintain other restricted progenitors or modulate HSC or niche function. Recent studies identified new markers that resolve some of the heterogeneity among stromal cells and refine the localization of restricted progenitor niches. Other recent studies identified ways in which niches regulate HSC function and hematopoiesis beyond growth factors. We summarize the current understanding of hematopoietic niches, review recent progress, and identify important unresolved questions.

Jarid2 Restricts Long-Term Repopulating Stem Cell Capacity in Multipotent Progenitors and Acts As Tumor Suppressor in Chronic Myeloid Neoplasms

Myeloid disorders including myeloproliferative neoplasms (MPN) are clonal diseases of hematopoietic stem or progenitor cells that arise from genetic and epigenetic alterations which perturb key processes such as self-renewal, proliferation and differentiation. The most common genetic mutations found in the MPN initiating clone occur in the genes JAK2, MPL and CALR, and these mutations are likely responsible for initiation of disease. Acquisition of additional co-operating mutations in genes such as TET2, IDH1/2, and NRAS in the MPN clone can give rise to secondary acute myeloid leukemia (sAML), a much more aggressive disease with an average survival time after transformation less than six months. Recent genome sequencing studies identified chromosomal deletions of JARID2, a Polycomb Repressive Complex 2 (PRC2) co-factor involved in implementing H3K27me3 marks, in the leukemic phase of post-MPN sAML but not in chronic MPN phase of the disease. These data indicate that JARID2 might act as a tumor suppressor in chronic myeloid disorders and genetic deletion of JARID2 could be a sAML-specific transforming event.To investigate the role of Jarid2 as a tumor suppressor, we utilized an inducible mouse model of the prototypical MPN driver mutation Jak2V617F. The absence of Jarid2 in a Jak2V617F/+ background accelerated MPN progression, with a median survival of 23 and 43 days post Jarid2 -deletion for Jarid2-/-- Jak2V617F/+ and Jarid2+/-- Jak2V617F/+ mice, respectively, compared to 113 days for Jak2V617F/+ control mice. Together, these data suggest that loss of Jarid2 in Jak2V617F/+ background accelerates MPN pathogenesis.To more completely understand the tumor suppressor role of Jarid2 in MPNs, we aimed to define its role in normal hematopoiesis. We deleted Jarid2 using the Vav-CRE driver to facilitate conditional inactivation of Jarid2 in hematopoietic system. Competitive transplantation was employed to assess the hematopoietic stem cell (HSC) potential. Either 100 phenotypically defined HSCs (Lineage-cKit+ScaI+CD48-CD150+) or 2.5x105 whole bone marrow (WBM) cells were transplanted into lethally irradiated recipient mice along with 2.5 x 105 WBM cells from genetically distinguishable WT mice. At 16 weeks post-transplant, the analysis of test cell contribution in recipient mice revealed that Vav-CRE: Jarid2 -KO HSCs did not contribute to peripheral blood (PB) chimerism. In contrast, Vav-CRE: Jarid2 -KO WBM contributed to PB lineages at levels comparable to control WBM. These data suggested that a population of cell(s) outside phenotypically-defined HSCs contribute(s) to hematopoiesis in the absence of Jarid2 . To find the population(s) that reside in Vav-CRE: Jarid2 -KO WBM with long-term multi-lineage capacity, we transplanted the following sorted progenitor populations: multipotent progenitors (50 MPPs; Lineage-cKit+ScaI+CD48-CD150-) and restricted progenitors (250 RPs; Lineage -cKit+ScaI+CD48+CD150-) in a competitive setting as described above. As expected, neither control MPPs or RPs were capable of long-term multi-lineage reconstitution. Strikingly, Vav-CRE: Jarid2 -KO MPPs engrafted in primary and secondary recipients, generating tri-lineage hematopoiesis effectively, whereas Vav-CRE: Jarid2 -KO RPs did not engraft long-term in any recipients. Additionally, MPPs isolated from Jak2V617F - Jarid2KO mice can propagate MPN in transplanted recipients, unlike MPPs transplanted from Jak2V617F mice. RNA-seq expression profiling revealed upregulation of Runx1t1 and Mycn in Jarid2-KO MPPs, genes which have been used to reprogram committed blood cells to a multipotent state. We hypothesized that the inability to silence Runx1t1 and Mycn in Jarid2-KO MPPs was a mechanism that permits self-renewal in this normally transiently-repopulating cell fraction. Enforced expression of Runx1t1 and Mycn in WT MPPs endowed long-term repopulating capacity in vivo, phenocopying the behavior of Jarid2-KO MPPs. Cumulatively, our data suggest that the normal role of Jarid2 in hematopoiesis is to restrict self-renewal capacity to long-term HSCs, and that dysregulation of this process can lead to hematopoietic malignancies. DisclosuresNo relevant conflicts of interest to declare.

MLL3 Deletion Enhances HSC Self-Renewal

Mixed lineage leukemia 3 (MLL3 ) is one of the most commonly mutated genes in human cancer, and it is believed to function as a tumor suppressor. It is one of many genes affected as a result of mono-allelic 7q deletions that occur commonly in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MLL3 encodes a histone methyltransferase that catalyzes the monomethylation of Histone 3 lysine 4 (H3K4), a known feature of enhancer elements. MLL3 also nucleates a COMPASS-like complex that removes repressive H3K27me3 marks, recruits the histone acetyltransferase p300/CBP to enhancers and promotes transcript elongation at promoters. For these reasons, MLL3 binding at cis-regulatory elements is thought to potentiate enhancer activity and transcription. Previous studies have shown that Mll3 deficiency causes a mild myeloproliferative disorder in mice (PLoS ONE 11:e0162515), and shRNA knockdown of Mll3 has been shown to cooperate with loss of Nf1 and p53 to promote AML (Cancer Cell 25:652). In the latter study, Mll3 silencing was associated with a hematopoietic stem cell (HSC)-like gene expression signature within the AML cells. These studies implicate MLL3 as a hematopoietic tumor suppressor, though its precise function in HSC self-renewal and lineage commitment has not been described.To better understand how Mll3 regulates HSC self-renewal, we developed a novel loss-of-function mouse allele in which a 5-base pair deletion and frameshift mutation was created near the N-terminus of the protein. Homozygous Mll3-/- mice are viable through most of gestation, but they die just prior to birth. Heterozygous Mll3+/- mice are born at normal Mendelian ratios, their development is normal after birth and they are fertile. To test whether Mll3 regulates hematopoiesis, we determined the frequencies and absolute numbers of HSCs, lineage restricted hematopoietic progenitors (HPCs), and granulocyte-monocyte progenitors (GMPs) in control, Mll3+/- and Mll3-/- fetal livers (E18.5) and control and Mll3+/- adult bone marrow. Heterozygous and homozygous Mll3 deletion caused a modest increase in HSC numbers and a modest decrease in HPC frequency in fetal livers. Mll3 heterozygosity resulted in a significant expansion of the HSC population in adult bone marrow. To test whether these increases in HSC numbers reflected enhanced self-renewal, we performed serial transplantation assays with fetal and adult HSCs. While primary transplants showed only a modest increase in repopulating activity in Mll3+/- and Mll3-/- HSCs, this effect was greatly enhanced in secondary transplants. Thus, Mll3 deficiency enhances HSC self-renewal capacity. Despite this phenotype, RNAseq showed no differences in the transcriptomes of wild type and Mll3+/- HSCs or HPCs. This suggests that Mll3 loss may reduce the probability of lineage commitment during successive HSC divisions without altering the commitment pathways themselves. Ongoing experiments will test this hypothesis. Enhanced self-renewal, particularly under conditions of hematopoietic stress, may convey a selective advantage to Mll3 -heterozygous HSCs and enhance progression to MDS. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial Carrier Homolog 2 (MTCH2) Regulates the Differentiation of AML Cells By Controlling Pyruvate Entry into the Mitochondria, Nuclear Localization of Pyruvate Dehydrogenase Complex and H3 and H4 Histone Acetylation

Mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein that functions as a receptor for pro-apoptotic BID. MTCH2 also regulates metabolism through undefined mechanisms.We identified MTCH2 as essential for AML growth and viability in a CRISPR screen. OCI-AML2 leukemia cells overexpressing CAS9 were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors. We focused on the gRNA that targeted the 1050 mitochondrial proteins and identified MTCH2 within the top 1% of hits. We validated this finding using two individual sgRNAs, where knockout of MTCH2 reduced the growth and viability of CAS9-OCI-AML2 cells by >75%. Additionally, knockdown of MTCH2 with shRNA reduced growth and viability of OCI-AML2 (>90%), TEX (>80%), U937 (>65%), and HL60 (>75%) leukemia cells. Knockdown of MTCH2 targeted the leukemia initiating cells as it abrogated engraftment of TEX cells into immune deficient mice. We also infected hematopoietic cells from wild type and MTCH2 flox/flox Vav1-cre mice with the MLL-AF9 oncogene. Knockout of MTCH2 decreased the leukemogenic potential of these cells and increased survival of the mice.In contrast, knockout of MTCH2 has more subtle effects on normal murine hematopoiesis. flox/flox Vav1-cre MTCH2 knockout mice had normal peripheral blood counts, but a ~15% reduction in the abundance of hematopoietic stem cells with a slight increase in the restricted progenitor populations (Maryanovich et al, Nat Comm 2015).Murine hematopoietic cells from MTCH2 -/- mice have increased mitochondrial mass and increased oxidative phosphorylation. Yet, no changes in basal oxygen consumption, mitochondrial ROS, or respiratory chain activity were seen in AML cells after MTCH2 knockdown.We examined changes in gene expression by RNA-sequencing in OCI-AML-2 cells after MTCH2 knockdown. MTCH2 knockdown increased expression of genes involved in cellular differentiation and decreased genes that maintain stemness. Therefore, we analyzed differentiation of AML cells after MTCH2 knockdown and demonstrated increased non-specific esterase staining and increased expression of CD cell surface markers associated with differentiation.In lung cancer mitochondrial stress induces the translocation of pyruvate dehydrogenase (PDH) from the mitochondria to the nucleus, where it increases conversion of pyruvate to Acetyl CoA which, in turn, acetylates histones. Therefore, we examined changes in PDH localization and histone acetylation after MTCH2 knockdown in OCI-AML-2 and TEX cells. MTCH2 knockdown decreased mitochondrial pyruvate, increased nuclear localization of pyruvate dehydrogenase complex (PDH) and increased acetylation of H3 and H4 histones. Over-expression of the PDH E2 subunit with a nuclear localizing signal mimicked MTCH2 knockdown and decreased cell growth, induced differentiation, and increased H3 and H4 histone acetylation in. Likewise, the acetyltransferase inhibitor, C646, abrogated the effects of MTCH2 knockdown on histone acetylation, growth and differentiation. We also demonstrated that knockdown of MTCH2 decreased levels of mitochondrial pyruvate in OCI-AML-2 cells.The entry of pyruvate into the mitochondria is regulated by the Mitochondrial Pyruvate Carrier (MPC). Therefore, we treated AML cells (OCI-AML2, TEX, HL60, and 8227) with the selective MPC inhibitor UK5099. UK5099 mimicked the effects of MTCH2 knockdown and decreased mitochondrial levels of pyruvate, reduced cell growth and viability, increased levels of acetylated histones, increased nuclear levels of PDH and promoted differentiation of AML cells. Treatment of mice with UK5099 (40mg/kg) decreased growth of OCI-AML2 xenografts. Finally, UK5099 induced cell death by Annexin V/PI staining in undifferentiated primary AML cells, but differentiated primary AML cells (by morphology and CD marker staining) and normal hematopoietic cells were more resistant.In summary, inhibiting entry of pyruvate into the mitochondria is a new mechanism to regulate differentiation of AML cells and stem cells by increasing PDH localization to the nucleus and histone acetylation. Thus, MTCH2 or MPC inhibitors may be novel therapeutics agents for AML. DisclosuresSchimmer:Medivir: Research Funding; Takeda Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Early cis-regulatory events in the formation of retinal horizontal cells

During retinal development, multipotent and restricted progenitor cells generate all of the neuronal cells of the retina. Among these are horizontal cells, which are interneurons that modulate the light-induced signal from photoreceptors. This study utilizes the identification of novel cis-regulatory elements as a method to examine the gene regulatory networks that direct the development of horizontal cells. Here we describe a screen for cis-regulatory elements, or enhancers, for the horizontal cell-associated genes PTF1A, ONECUT1 (OC1), TFAP2A (AP2A), and LHX1. The OC1ECR22 and Tfap2aACR5 elements were shown to be potential enhancers for OC1 and TFAP2A, respectively, and to be specifically active in developing horizontal cells. The OC1ECR22 element is activated by PTF1A and RBPJ, which translates to regulation of OC1 expression and suggests that PTF1A is a direct activator of OC1 expression in developing horizontal cells. The region within the Tfap2aACR5 element that is responsible for its activation was determined to be a 100 bp sequence named Motif 4. Both OC1ECR22 and Tfap2aACR5 are negatively regulated by the nuclear receptors THRB and RXRG, as is the expression of OC1 and AP2A, suggesting that nuclear receptors may have a role in the negative regulation of horizontal cell development.

Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs.