Maintenance Of Adult Stem Cells Research Articles

Red2Flpe-SCON: a versatile, multicolor strategy for generating mosaic conditional knockout mice

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.

The ATF4-RPS19BP1 axis modulates ribosome biogenesis to promote erythropoiesis

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

Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway

BackgroundXerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation.MethodsObtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro.ResultshMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation.ConclusionsWe showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

High Intrinsic Oncogenic Potential in the Myc-Box-Deficient Hydra Myc3 Protein.

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver.

Notch signaling sculpts the stem cell niche.

Adult stem cells depend on their niches for regulatory signaling that controls their maintenance, division, and their progeny differentiation. While communication between various types of stem cells and their niches is becoming clearer, the process of stem cell niche establishment is still not very well understood. Model genetic organisms provide simplified systems to address various complex questions, for example, how is a stem cell niche formed? What signaling cascades induce the stem cell niche formation? Are the mechanisms of stem cell niche formation conserved? Notch signaling is an evolutionarily conserved pathway first identified in fruit flies, crucial in fate acquisition and spatiotemporal patterning. While the core logic behind its activity is fairly simple and requires direct cell-cell interaction, it reaches an astonishing complexity and versatility by combining its different modes of action. Subtleties such as equivalency between communicating cells, their physical distance, receptor and ligand processing, and endocytosis can have an effect on the way the events unfold, and this review explores some important general mechanisms of action, later on focusing on its involvement in stem cell niche formation. First, looking at invertebrates, we will examine how Notch signaling induces the formation of germline stem cell niche in male and female Drosophila. In the developing testis, a group of somatic gonadal precursor cells receive Delta signals from the gut, activating Notch signaling and sealing their fate as niche cells even before larval hatching. Meanwhile, the ovarian germline stem cell niche is built later during late larval stages and requires a two-step process that involves terminal filament formation and cap cell specification. Intriguingly, double security mechanisms of Notch signaling activation coordinated by the soma or the germline control both steps to ensure the robustness of niche assembly. Second, in the vast domains of mammalian cellular signaling, there is an emerging picture of Notch being an active player in a variety of tissues in health and disease. Notch involvement has been shown in stem cell niche establishment in multiple organs, including the brain, muscle, and intestine, where the stem cell niches are essential for the maintenance of adult stem cells. But adult stem cells are not the only cells looking for a home. Cancer stem cells use Notch signaling at specific stages to gain an advantage over endogenous tissue and overpower it, at the same time acquiring migratory and invasive abilities to claim new tissues (e.g., bone) as their territory. Moreover, in vitro models such as organoids reveal similar Notch employment when it comes to the developing stem cell niches. Therefore, a better understanding of the processes regulating stem cell niche assembly is key for the fields of stem cell biology and regenerative medicines.

The mitochondrial protein OPA1 regulates the quiescent state of adult muscle stem cells.

Quiescence regulation is essential for adult stem cell maintenance and sustained regeneration. Our studies uncovered that physiological changes in mitochondrial shape regulate the quiescent state of adult muscle stem cells (MuSCs). We show that MuSC mitochondria rapidly fragment upon an activation stimulus, via systemic HGF/mTOR, to drive the exit from deep quiescence. Deletion of the mitochondrial fusion protein OPA1 and mitochondrial fragmentation transitions MuSCs into G-alert quiescence, causing premature activation and depletion upon a stimulus. OPA1 loss activates a glutathione (GSH)-redox signaling pathway promoting cell-cycle progression, myogenic gene expression, and commitment. MuSCs with chronic OPA1 loss, leading to mitochondrial dysfunction, continue to reside in G-alert but acquire severe cell-cycle defects. Additionally, we provide evidence that OPA1 decline and impaired mitochondrial dynamics contribute to age-related MuSC dysfunction. These findings reveal a fundamental role for OPA1 and mitochondrial dynamics in establishing the quiescent state and activation potential of adult stem cells.

The RNA-binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance.

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here, we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing is critical for Drosophila melanogaster intestinal stem cells and their daughter cells, enteroblasts, to maintain their progenitor cell properties and functions. Loss of swm causes intestinal stem cells and enteroblasts to stop dividing and instead detach from the basement membrane, resulting in severe progenitor cell loss. swm loss is further characterized by nuclear accumulation of poly(A)+ RNA in progenitor cells. Second Mitotic Wave Missing associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Second Mitotic Wave Missing-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly ortholog of Filamin. Taken together, this study indicates a nuclear role for Second Mitotic Wave Missing in adult stem cell maintenance, raising the possibility that nuclear post-transcriptional regulation of mRNAs encoding cell adhesion proteins ensures proper attachment of progenitor cells.

Abstract 5574: In vivo characterization of a novel CAR-T cell therapy directed towards LGR5 for the treatment of colorectal cancer

Abstract Colorectal cancer (CRC) is the third most diagnosed cancer and incidence rates are rapidly increasing among young adults. Despite current treatments, the 5-year survival rate is approximately 65%, highlighting an urgent need for new therapies. Leucine-rich repeat-containing G-protein coupled receptor (LGR5) is a promising antigen target for chimeric antigen receptor (CAR)-T cell therapy, which may represent a novel effective treatment for CRC. LGR5 binds R-spondins and functions to enhance Wnt and β-catenin signalling involved in maintenance and proliferation of adult stem cells. Expression of LGR5 has been previously reported on cancer stem cells in CRC, and has been demonstrated to play a crucial role in CRC tumor initiation and metastasis. Flow cytometric analysis of LGR5 cell surface expression on in vitro and ex vivo human CRC tumour cells revealed a subpopulation of LGR5+ cells. Six LGR5-targetting CD3+ CAR-T cell variants were developed, which differed in CAR variable heavy (VH) and variable light (VL) chain orientation and hinge length. As preliminary experimentation identified that four of the six CAR-T cell variants displayed robust anti-tumour cytotoxicity in vitro, these CAR-T cell variants were tested for in vivo anti-tumour efficacy using a subcutaneous human colorectal tumor xenograft mouse model. Pre-injection phenotyping after freeze-thawing of the CAR-T cells revealed greater than 94% transduction efficiency, low expression of co-inhibitory receptors CTLA-4 and PD-1 and a high frequency of central memory T cells (CD62L+ CD45RO+). Additionally, a high frequency of the CD8+ compartment of these CAR-T cells expressed perforin, CD107a, Granzyme B and TNFα. For each variant, intravenous injection of 20x106 LGR5-targeting CAR-T cells induced significant tumor regression and gave 100-day survival post-tumor injection. Ex vivo analyses of tumors and spleens via flow cytometry at endpoint indicated CAR-T cell persistence to 100-days post-tumor injection. Of the CAR-T cells isolated from tumours ex vivo, high level expression of co-inhibitory receptors PD-1, CTLA-4, and TIM-3 was observed. The splenic T cell population was enriched for central memory T cells and expressed high levels of PD-1. Complete tumor remission was achieved in 100% of mice receiving the VH-VL orientation, medium hinge CAR construct (designated CNA3103), prompting further investigation of this variant. Intravenous injection of 5x106 CNA3103 CAR-T cells was sufficient to induce tumour remission and 100-day survival. Similarly, complete tumour rejection was achieved with a dose of 2.5x106 CNA3103 CAR-T cells in a 30-day in vivo experiment. These results demonstrate that LGR5-targeting CAR-T cells exhibit potent in vivo anti-tumor efficacy and position LGR5 as a promising CAR target antigen, prompting investigation of LGR5-targeting CAR-T cells as a therapeutic for CRC in future clinical trials. Citation Format: Dylan J. McPeake, Timona S. Tyllis, Jade Foeng, Veronika Bandara, Caitlin A. Abbott, Batjargal Gundsambuu, Elaheh Rohani-Rad, Silvana Napoli, Timothy Sadlon, Simon C. Barry, Shaun R. McColl. In vivo characterization of a novel CAR-T cell therapy directed towards LGR5 for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5574.

MLL1 is required for maintenance of intestinal stem cells.

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

SETD5 modulates homeostasis of hematopoietic stem cells by mediating RNA Polymerase II pausing in cooperation with HCF-1

SETD5 mutations were identified as the genetic causes of neurodevelopmental disorders. While the whole-body knockout of Setd5 in mice leads to embryonic lethality, the role of SETD5 in adult stem cell remains unexplored. Here, a critical role of Setd5 in hematopoietic stem cells (HSCs) is identified. Specific deletion of Setd5 in hematopoietic system significantly increased the number of immunophenotypic HSCs by promoting HSC proliferation. Setd5-deficient HSCs exhibited impaired long-term self-renewal capacity and multiple-lineage differentiation potentials under transplantation pressure. Transcriptome analysis of Setd5-deficient HSCs revealed a disruption of quiescence state of long-term HSCs, a cause of the exhaustion of functional HSCs. Mechanistically, SETD5 was shown to regulate HSC quiescence by mediating the release of promoter-proximal paused RNA polymerase II (Pol II) on E2F targets in cooperation with HCF-1 and PAF1 complex. Taken together, these findings reveal an essential role of SETD5 in regulating Pol II pausing-mediated maintenance of adult stem cells.

Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man.

Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases in the cytosol. In the recent two decades, a plethora of studies established that elevated ROS levels generated by oxidative eustress are crucial physiological mediators of many cellular and developmental processes. In this review, we discuss the mechanisms of ROS generation and regulation, current understanding of ROS functions in the maintenance of adult and embryonic stem cells, as well as in the process of cell reprogramming to a pluripotent state. Recently discovered cell-non-autonomous ROS functions mediated by growth factors are crucial for controlling cell differentiation and cellular immune response in Drosophila. Importantly, many physiological functions of ROS discovered in Drosophila may allow for deciphering and understanding analogous processes in human, which could potentially lead to the development of novel therapeutic approaches in ROS-associated diseases treatment.

Effect of the Wnt/β-catenin pathway inhibitors on cell proliferation and migration of HEC-1A endometrial adenocarcinoma: experimental cell culture model

Objectives: The most diagnosed tumor among infiltrating tumors of the female genital tract is endometrial carcinoma. The Wnt/β-catenin signaling pathway has an important role in organogenesis, self-renewal of tissues, and adult stem cell maintenance. However aberrant activation of it causes many types of tumors and also related to the prognosis of patients. Therefore, we aimed to investigate whether Wnt/β-catenin pathway inhibitors have any effect on the proliferation and migration of tumor cells. Methods: As cancer cell line, HEC-1A endometrial adenocarcinoma was used. The Wnt/β-catenin pathway inhibitors effects on proliferation and migration were demonstrated by real-time cell analysis device and wound healing model respectively. Results: Wnt/β-catenin pathway inhibitors FH535 (25 μM at 36th hour, p < 0.05; 50μM at 48th hour p < 0.001) and niclosamide inhibited cell proliferation (10, 25 and 50μM at 60th hours; p < 0.01, p < 0.05 and p < 0.001, respectively) whereas ICRT14 and IWP-2 did not. However only niclosamide which is also an antihelmintic drug inhibited migration of the cells in all concentrations tested (10, 25 and 50μM, p < 0.05). Conclusions: The present study shows that the Wnt/β-catenin pathway has an substantial role in both proliferation and migration of endometrial adenocarcinoma. We suggest that the antihelmintic drug niclosamide could be further investigated for its potential therapeutic effect in endometrial adenocarcinoma.

Lipids and Hedgehogs

The Hedgehog cell-cell signaling pathway governs critical processes in the embryonic development of most animals, in adult stem cell maintenance, and in cancer. The pathway is activated by the secreted Hedgehog morphogen, such as the prototypical vertebrate homologue, Sonic hedgehog (SHH). SHH is generated in the endoplasmic reticulum of signal-producing cells, as a hydrophobic protein uniquely modified with palmitate at the N-terminus and with cholesterol at the C-terminus. These lipid modifications, which are critical for SHH function, render SHH strongly attached to the plasma membrane; yet the SHH morphogen must travel through the extracellular space, to reach responding cells located far away. I will present our recent work aimed at elucidating the biochemical mechanisms that allow the lipidated SHH morphogen to be released in diffusible form from producing cells, and then be delivered to its receptor, the tumor suppressor membrane protein Patched1, on the surface of responding cells.

Zfp521 is essential for the quiescence and maintenance of adult hematopoietic stem cells under stress

SummaryZinc finger protein 521 (Zfp521), a quiescent hematopoietic stem cell (HSC)-enriched transcription factor, is involved in the self-renewal and differentiation of fetal liver HSC. However, its role in adult hematopoiesis remains elusive. Here, we found that Zfp521 deletion did not inhibit adult hematopoiesis under homeostatic conditions. In contrast, Zfp521-null chimeric mice showed significantly reduced pool size of HSC and hematopoietic progenitor cells associated with increased apoptosis and loss of quiescence. Competitive serial transplantation assays revealed that Zfp521 regulates HSC self-renewal and differentiation under regenerative stress. Mechanistically, Zfp521 transcriptionally repressed Rela expression by increasing H3K9ac and decreasing H3K9me3 levels in its promoter. Knockdown of Rela inhibited the hyper-activated NF-κB pathway and reversed the loss of quiescence in Zfp521-null HSC under stress. Thus, our results reveal a previously unrecognized role for Zfp521 as critical regulator of quiescence and self-renewal of HSC in adult hematopoiesis mediated at least partly by controlling Rela expression.

Prdm16 is a critical regulator of adult long-term hematopoietic stem cell quiescence

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene Prdm16 during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. To investigate the role of Prdm16 in adult HSCs, we generated a novel conditional knockout mouse model and deleted Prdm16 in adult mouse hematopoietic system using the IFN-inducible Mx1-Cre Our results show that Prdm16 deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of Prdm16 significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which Cdkn1a and Egr1 were identified as direct targets of Prdm16 Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence.

Combination of SB431542, Chir9901, and Bpv as a novel supplement in the culture of umbilical cord blood hematopoietic stem cells

BackgroundSmall molecule compounds have been well recognized for their promising power in the generation, expansion, and maintenance of embryonic or adult stem cells. The aim of this study was to identify a novel combination of small molecules in order to optimize the ex vivo expansion of umbilical cord blood-derived CD34+ cells.MethodsConsidering the most important signaling pathways involved in the self-renewal of hematopoietic stem cells, CB-CD34+ cells were expanded with cytokines in the presence of seven small molecules including SB, PD, Chir, Bpv, Pur, Pμ, and NAM. The eliminativism approach was used to find the best combination of selected small molecules for effective ex vivo expansion of CD34+ cell. In each step, proliferation, self-renewal, and clonogenic potential of the expanded cells as well as expression of some hematopoietic stem cell-related genes were studied. Finally, the engraftment potential of expanded cells was also examined by the mouse intra-uterine transplantation model.ResultsOur data shows that the simultaneous use of SB431542 (TGF-β inhibitor), Chir9901 (GSK3 inhibitor), and Bpv (PTEN inhibitor) resulted in a 50-fold increase in the number of CD34+CD38− cells. This was further reflected in approximately 3 times the increase in the clonogenic potential of the small molecule cocktail-expanded cells. These cells, also, showed a 1.5-fold higher engraftment potential in the peripheral blood of the NMRI model of in utero transplantation. These results are in total conformity with the upregulation of HOXB4, GATA2, and CD34 marker gene as well as the CXCR4 homing gene.ConclusionTaken together, our findings introduce a novel combination of small molecules to improve the yield of existing protocols used in the expansion of hematopoietic stem cells.

Prdm16 Is a Critical Regulator of Adult Long-Term Hematopoietic Stem Cell Quiescence

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). PRDM16 encodes a zinc-finger transcription factor homologous to MECOM and was first cloned from chromosome translocations in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Disruption of Prdm16 during mouse embryonic development has been shown to cause a profound loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. Using a novel Prdm16 conditional knockout mouse model, we show that Prdm16 deletion in adult mouse hematopoietic system have a less severe effect on HSCs, causing a gradual decline in adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-FU injections. Annexin V staining showed no difference in apoptosis between wild type and knockout adult long-term HSCs (LT-HSCs). In contrast, BrdU analysis revealed that loss of Prdm16 significantly increases cycling of LT-HSCs with majority of the cells found in the S to G2/M phase. Consistently, RNA-seq analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs. Two such genes were further identified as direct targets of Prdm16. Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence. Disclosures No relevant conflicts of interest to declare.

Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand.

Hedgehog signaling governs critical processes in embryogenesis, adult stem cell maintenance, and tumorigenesis. The activating ligand, Sonic hedgehog (SHH), is highly hydrophobic because of dual palmitate and cholesterol modification, and thus, its release from cells requires the secreted SCUBE proteins. We demonstrate that the soluble SCUBE-SHH complex, although highly potent in cellular assays, cannot directly signal through the SHH receptor, Patched1 (PTCH1). Rather, signaling by SCUBE-SHH requires a molecular relay mediated by the coreceptors CDON/BOC and GAS1, which relieves SHH inhibition by SCUBE. CDON/BOC bind both SCUBE and SHH, recruiting the complex to the cell surface. SHH is then handed off, in a dual lipid-dependent manner, to GAS1, and from GAS1 to PTCH1, initiating signaling. These results define an essential step in Hedgehog signaling, whereby coreceptors activate SHH by chaperoning it from a latent extracellular complex to its cell-surface receptor, and point to a broader paradigm of coreceptor function.

Lineage hierarchies and stochasticity ensure the long-term maintenance of adult neural stem cells.

The cellular basis and extent of neural stem cell (NSC) self-renewal in adult vertebrates, and their heterogeneity, remain controversial. To explore the functional behavior and dynamics of individual NSCs, we combined genetic lineage tracing, quantitative clonal analysis, intravital imaging, and global population assessments in the adult zebrafish telencephalon. Our results are compatible with a model where adult neurogenesis is organized in a hierarchy in which a subpopulation of deeply quiescent reservoir NSCs with long-term self-renewal potential generate, through asymmetric divisions, a pool of operational NSCs activating more frequently and taking stochastic fates biased toward neuronal differentiation. Our data further suggest the existence of an additional, upstream, progenitor population that supports the continuous generation of new reservoir NSCs, thus contributing to their overall expansion. Hence, we propose that the dynamics of vertebrate neurogenesis relies on a hierarchical organization where growth, self-renewal, and neurogenic functions are segregated between different NSC types.

Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc.

Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.